Amino acid sequences directed against il-6r and polypeptides comprising the same for the treatment of il-6r related diseases and disorders

ABSTRACT

The present invention relates to amino acid sequences that are directed against/and or that can specifically bind Interleukin-6 Receptor (IL-6R) with improved affinity and/or avidity, and/or that have an improved efficacy and/or potency, and which are capable of (partially, or preferably totally) blocking the IL-6/IL-6R interaction and/or inhibit signalization through IL-6, 1L-6R and/or the IL-6/IL-6R complex. The invention further relates to compounds or constructs, and in particular proteins and polypeptides, that comprise or essentially consist of one or more such amino acid sequences. The invention also relates to nucleic acids encoding such amino acid sequences and polypeptides, to methods for preparing such amino acid sequences and polypeptides, to host cells expressing or capable of expressing such amino acid sequences or polypeptides, to compositions, and in particular to pharmaceutical compositions, that comprise such amino acid sequences, polypeptides, nucleic acids and/or host cells, and to uses of such amino acid sequences or polypeptides, nucleic acids, host cells and/or compositions, in particular for prophylactic, therapeutic or diagnostic purposes.

FIELD OF THE INVENTION

The present invention relates to amino acid sequences that are directedagainst/and or that can specifically bind (as defined herein)Interleukin-6 Receptor (IL-6R), as well as to compounds or constructs,and in particular proteins and polypeptides, that comprise oressentially consist of one or more such amino acid sequences (alsoreferred to herein as “amino acid sequences of the invention”,“compounds of the invention”, “constructs of the invention” and“polypeptides of the invention”, respectively).

The invention also relates to nucleic acids encoding such amino acidsequences and polypeptides (also referred to herein as “nucleic acids ofthe invention” or “nucleotide sequences of the invention”); to methodsfor preparing such amino acid sequences and polypeptides; to host cellsexpressing or capable of expressing such amino acid sequences orpolypeptides; to compositions, and in particular to pharmaceuticalcompositions, that comprise such amino acid sequences, polypeptides,nucleic acids and/or host cells; and to uses of such amino acidsequences or polypeptides, nucleic acids, host cells and/orcompositions, in particular for prophylactic, therapeutic or diagnosticpurposes, such as the prophylactic, therapeutic or diagnostic purposesmentioned herein.

Other aspects, embodiments, advantages and applications of the inventionwill become clear from the further description herein.

BACKGROUND ART

The interaction of IL-6, a protein originally identified as a B celldifferentiation factor (Hirano et al., 1985, Proc. Natl. Acad. Sci. USA,82: 5490-4; EP 0257406), with IL-6R (Yamasaki et al., 1988, Science,241: 825-8; EP 0325474) results in the formation of the IL-6/IL-6Rcomplex. This complex binds to gp130 (Taga et al., 1989, Cell, 58:573-81; EP 0411946), a membrane protein on a target cell, whichtransmits various physiological actions of IL-6. IL-6 is currently knownto be involved in—amongst others—the regulation of the immune response,hematopoiesis, the acute phase response, bone metabolism, angiogenesis,and inflammation. Deregulation of IL-6 production is implicated in thepathology of several autoimmune and chronic inflammatory proliferativedisease processes (Ishihara and Hirano, 2002, Biochim. Biophys. Acta,1592: 281-96). As a consequence, inhibitors of IL-6 induced signalinghave attracted much attention in the past (Hirano et al., 1990, Immunol.Today, 11: 443-9). Polypeptides specifically binding to IL-6 (Klein etal., 1991, Blood, 78: 1198-204; EP 0312996), IL-6R (EP 0409607) or gp130(Saito et al., 1993, J. Immunol. Methods, 163: 217-223; EP 0572118)proved to exhibit an efficient inhibitory effect on IL-6 functioning.

IL-6 overproduction and signalling (and in particular so-calledtrans-signalling) are involved in various diseases and disorders, suchas sepsis (Starnes et al., 1999, J. Immunol., 148: 1968) and variousforms of cancer such as multiple myeloma disease (MM), renal cellcarcinoma (RCC), plasma cell leukaemia (Klein et al., 1991), lymphoma,B-lymphoproliferative disorder (BLPD) and prostate cancer. Non-limitingexamples of other diseases caused by excessive IL-6 production orsignalling include bone resorption (osteoporosis) (Roodman et al., 1992,J. Bone Miner. Res., 7: 475-8; Jilka et al., 1992, Science, 257: 88-91),cachexia (Strassman et al., 1992, J. Clin. Invest. 89: 1681-1684),psoriasis, mesangial proliferative glomerulonephritis, Kaposi's sarcoma,AIDS-related lymphoma (Emilie et al., 1994, Int. J. Immunopharmacol. 16:391-6), inflammatory diseases and disorder such as rheumatoid arthritis,systemic onset juvenile idiopathic arthritis, hypergammaglobulinemia(Grau at al., 1990, J. Exp. Med. 172: 1505-8); Crohn's disease,ulcerative colitis, systemic lupus erythematosus (SLE), multiplesclerosis, Castleman's disease, IgM gammopathy, cardiac myxoma, asthma(in particular allergic asthma) and autoimmune insulin-dependentdiabetes mellitus (Campbell et al., 1991, J. Clin. Invest. 87: 739-742).Other IL-6 related disorders will be clear to the skilled person.

As can for example be seen from the references above, the prior artdescribes antibodies and antibody fragments directed against human IL-6,against human IL-6R and against human gp130 protein for the preventionand treatment of IL-6 relates disorders. Examples are Tocilizumab (seeWoo et al., 2005, Arthritis Res. Ther. 7: 1281-8; Nishimoto et al.,2005, Blood 106: 2627-32; Ito et al., 2004, Gastroenterology, 126:989-96; Choy at al., 2002, Arthritis Rheum. 46: 3143-50), BE8 (seeBataille et al., 1995, Blood 86: 685-91; Emilie et al., 1994, Blood 84:2472-9; Beck et al., 1994, N. Engl. J. Med. 330: 602-5; Wendling et al.,1993, J. Rheumatol. 20: 259-62) and CNTO-328 of Centocor (see Journal ofClinical Oncology, 2004, 22/14S: 2560; Journal of Clinical Oncology,2004, 22/145: 2608; Int. J. Cancer, 2004, 111:592-5). Another activeprinciple known in the art for the prevention and treatment of IL-6related disorders is an Fc fusion of soluble gp130 (see Becker et al.2004, Immunity, 21: 491-501; Doganci et al., 2005, J. Clin. Invest. 115:313-25; Nowell et al., 2003, J. Immunol. 171: 3202-9; Atreya et al.,2000, Nat. Med. 6: 583-8). Amino acid sequences and Nanobodies directedagainst IL-6R and polypeptides comprising the same are described in WO08/020,079.

SUMMARY OF THE INVENTION

A specific, but non-limiting object of the present invention is toprovide amino acid sequences, polypeptides and therapeutic compounds andcompositions that have improved therapeutic and/or pharmacologicalproperties, in addition to other advantageous properties (such as, forexample, improved ease of preparation and/or reduced costs of goods),compared to the prior art amino acid sequences, antibodies andNanobodies. These improved and advantageous properties will become clearfrom the further description herein. Without being limiting, the aminoacid sequences, polypeptides and therapeutic compounds and compositionsprovided by the invention may have an improved binding and/or affinity,improved avidity, improved efficacy and/or potency, an increasedselectivity and/or they may be capable of partially or preferablytotally blocking the IL-6/IL-6R interaction, and/or inhibitsignalization through IL-6, IL-6R and/or the IL-6/IL-6R complex.

Generally, it is an object of the invention to provide pharmacologicallyactive agents, as well as compositions comprising the same, that can beused in the diagnosis, prevention and/or treatment of one or more IL-6Rrelated disorders (as defined herein); and to provide methods for thediagnosis, prevention and/or treatment of such diseases and/or disordersthat involve the administration and/or use of such agents andcompositions.

The present invention relates to amino acid sequences (also referred toas “amino acid sequence(s) of the invention”) that are directedagainst/and or that can specifically bind (as defined herein)Interleukin-6 Receptor (IL-6R) with improved affinity and/or avidity,and/or that have an improved efficacy and/or potency, and which arecapable of (partially, or preferably totally) blocking the IL-6/IL-6Rinteraction and/or inhibit signalization through IL-6, IL-6R and/or theIL-6/IL-6R complex. More particularly, the present invention providesamino acid sequences that comprise one or more stretches of amino acidresidues chosen from the following:

-   -   a) SEQ ID NO's: 80-82; or    -   b) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with one of SEQ        ID NO's: 80-82, provided that the amino acid sequence comprising        said stretch of amino acid residues binds IL-6R with the same,        about the same, or a higher affinity compared to the amino acid        sequence comprising said stretch of amino acid residues without        the 2 or 1 amino acid difference, said affinity as measured by        surface plasmon resonance;        and/or    -   c) SEQ ID NO's: 84-91; or    -   d) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with one of SEQ        ID NO's: 84-91, provided that the amino acid sequence comprising        said stretch of amino acid residues binds IL-6R with the same,        about the same, or a higher affinity compared to the amino acid        sequence comprising said stretch of amino acid residues without        the 2 or 1 amino acid difference, said affinity as measured by        surface plasmon resonance;        and/or    -   e) SEQ ID NO's: 93-95; or    -   f) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with one of SEQ        ID NO's: 93-95, provided that the amino acid sequence comprising        said stretch of amino acid residues binds IL-6R with the same,        about the same, or a higher affinity compared to the amino acid        sequence comprising said stretch of amino acid residues without        the 2 or 1 amino acid difference, said affinity as measured by        surface plasmon resonance.

For binding to its epitope on IL-6R, an amino acid sequence will usuallycontain within its amino acid sequence one or more amino acid residuesor one or more stretches of amino acid residues (as further definedherein; i.e. with each “stretch” comprising two or more amino acidresidues that are adjacent to each other or in close proximity to eachother, i.e. in the primary or tertiary structure of the amino acidsequence) via which the amino acid sequence of the invention can bind tothe epitope on IL-6R. These amino acid residues or stretches of aminoacid residues thus form the “site” for binding to the epitope on IL-6R(also referred to herein as the “antigen binding site”; as furtherdefined herein).

The present invention provides a number of stretches of amino acidresidues (as defined herein) that are particularly suited for binding toa specific epitope on IL-6R. These stretches of amino acid residues maybe present in, and/or may be incorporated into, an amino acid sequenceof the invention, in particular in such a way that they form (part of)the antigen binding site of the amino acid sequence of the invention. Assuch, the resulting amino acid sequences, bind a specific epitope onIL-6R that lies in, forms part of, or overlaps with (i.e. in the primaryor tertiary structure) or is in close proximity to (i.e. in the primaryor tertiary structure) the IL-6 binding site on IL-6R (for example,competitively with IL-6); and as such, the resulting amino acidsequences are capable of partially or preferably totally blocking theIL-6/IL-6R interaction and/or inhibit signalization through IL-6, IL-6Rand/or the IL-6/IL-6R complex. In this context, the amino acid sequencesand polypeptides of the invention are preferably such that they cancompete with IL-6 for binding to the IL-6 receptor. The amino acidsequences and polypeptides of the invention are preferably such thatthey can compete for binding to the IL-6 receptor with the commerciallyavailable human-mouse reconstituted chimeric monoclonal anti-IL-6Rantibody Tocilizumab (MRA) (Chugai/Roche) or an antigen binding fragmentthereof (see for example WO 92/19759 and corresponding European patentEP 0628639, as well as Shinkura et al., 1998, Anticancer Research 18,1217-1222), for example in the assay described in Example 11; and/orsuch that they can bind to the same epitope or binding site on IL-6R asTocilizumab, or to an epitope close to said binding site and/oroverlapping with said binding site.

Also, the amino acid sequences of the invention are preferably such thatthey can compete for binding to the IL-6 receptor with the reference IgGas defined by SEQ ID NO's: 1 and 2 and/or the reference Fab as definedby SEQ ID NO's: 3 and 4 (see Example 1); and/or such that they can bindto the same epitope or binding site on IL-6R as said reference IgG orsaid reference Fab, or to an epitope close to said binding site and/oroverlapping with said binding site. For the preparation and sequence ofsaid reference IgG and reference Fab, reference is made to Example 1below, as well as to SEQ ID NO's: 1 to 4.

It should be noted that the invention in its broadest sense is notlimited to a specific structural role or function that these stretchesof amino acid residues may have in the amino acid sequence of theinvention, as long as these stretches of amino acid residues allow theamino acid sequence of the invention to bind to the specific epitope onIL-6R with a certain affinity and/or potency (as further definedherein). Thus, generally, the invention in its broadest sense comprisesany amino acid sequence that is capable of binding to the specificepitope on IL-6R and that comprises one or more stretches of amino acidresidues as described herein (and in particular a suitable combinationof two or more such stretches of amino acid residues) that are suitablylinked to each other via one or more further amino add sequences, suchthat the entire amino acid sequence forms a binding domain and/orbinding unit that is capable of binding to the specific epitope onIL-6R. It should however also be noted that the presence of only onesuch stretches of amino acid residues in an amino acid sequence of theinvention may by itself already be sufficient to provide an amino acidsequence of the invention that is capable of binding to the specificepitope on IL-6R (reference is for example again made to the so-called“Expedite fragments” described in WO 03/050531).

Amino acid sequences comprising one or more of these specific stretchesof amino acid residues show improved properties such as e.g. improvedbinding and/or affinity, improved avidity, improved efficacy andpotency, and/or an increased selectivity, in addition to their capacityto partially or totally block the IL-6/IL-6R interaction, and/or inhibitsignalization through IL-6, IL-6R and/or the IL-6/IL-6R complex.

More in particular, the amino acid sequences of the invention comprisingone or more of these specific stretches of amino acid residues can bindto IL-6R with an affinity (suitably measured and/or expressed as aK_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), ak_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, asfurther described herein) preferably such that they:

-   -   bind to hIL-6R with a dissociation constant (K_(D)) of 1 nM to 1        pM or less, preferably 500 pM to 1 pM or less, more preferably        100 pM to 1 pM or less, or even more preferably about 50 pM to 1        pM or less;        and/or such that they:    -   bind to cyno IL-6R with a dissociation constant (K_(D)) of 1 nM        to 1 pM or less, preferably 500 pM to 1 pM or less, more        preferably 100 pM to 1 pM or less, or even more preferably about        50 pM to 1 pM or less;        and/or such that they:    -   bind to hIL-61R with a k_(on)-rate of between 10⁴ M⁻¹s⁻¹ to        about 10⁷ M⁻¹s⁻¹, preferably between 10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹,        more preferably about 10⁶ M⁻¹s⁻¹ or more;        and/or such that they:    -   bind to cyno IL-6R with a k_(on)-rate of between 10⁴ M⁻¹s⁻¹ to        about 10⁷ M⁻¹s⁻¹, preferably between 10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹,        more preferably about 10⁶ M⁻¹s⁻¹ or more;        and/or such that they:    -   bind to hIL-6R with a k_(off) rate between 10⁻³ s⁻¹        (t_(1/2)=0.69 s) and 10⁻⁶ s⁻¹ (providing a near irreversible        complex with a t_(1/2) of multiple days), preferably between        10⁻⁴ s⁻¹ and 10⁻⁶s⁻¹, more preferably between 10⁻⁵ s⁻¹ and 10⁻⁶        s⁻¹, such as about 10⁻⁵ s⁻¹ or lower;        and/or such that they:    -   bind to cyno IL-6R with a k_(off) rate between 10⁻³ s⁻¹        (t_(1/2)=0.69 s) and 10⁻⁶ s⁻¹ (providing a near irreversible        complex with a t_(1/2) of multiple days), preferably between        10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹, more preferably between 10⁻⁵ s⁻¹ and 10⁻⁶        s⁻¹, such as about 10⁻⁵ s⁻¹ or lower.

Some preferred IC50 values for binding of the amino acid sequences ofthe invention to IL-6R will become clear from the further descriptionand examples herein.

For example, in the TF-1 assay as described by Kitamura et al. (1989, J.Cell Physiol., 140: 323), the amino acid sequences of the invention mayhave IC50 values (at 100 IU/mL IL-6) between 10 nM and 50 pM, preferablybetween 5 nM and 50 pM, more preferably between 1 nM and 50 pM or less,such as about 750 or 500 pM or less. In this TF-1 assay the amino acidsequences of the invention may have 1050 values (at 5000 IU/mL IL-6)between 50 nM and 1 nM, preferably between 25 nM and 1 nM, morepreferably between 10 nM and 1 nM or less, such as about 8 nM or less.In this TF-1 assay, the amino acid sequences of the invention may haveIC50 values that are at least the same and preferably better, at leasttwo times, preferably three times, more preferably four times, even morepreferably 5 times, 7 times or more than 7 times better compared to theIC50 value obtained for the reference IgG as defined by SEQ ID NO's: 1and 2 or the reference Fab as defined by SEQ ID NO's: 3 and 4 (seeExample 1). In this TF-1 assay, the amino acid sequences of theinvention may have IC50 values that are at least the same and preferablybetter, at least two times, preferably three times, more preferably fourtimes, even more preferably 5 times, 7 times or more than 7 times bettercompared to the IC50 value obtained for Tocilizumab (MRA).

In a plasma potency assay at EC50 values of IL-6 (e.g. in the presenceof 27.29 ng/mL IL-6 as described in Example 45), the amino acidsequences of the invention may have IC50 values between 500 pM and 50pM, preferably between 250 pM and 50 pM, more preferably between 200 pMand 50 pM or less, such as 150 pM or less. In a plasma potency assay atEC95 values of IL-6 (e.g. in the presence of 885 ng/mL IL-6 as describedin Example 45) the amino acid sequences of the invention may have IC50values between 1000 pM and 100 pM, preferably between 750 pM and 100 pM,more preferably between 500 pM and 100 pM or less, such as 400 pM orless. In this plasma potency assay, the amino acid sequences of theinvention may have IC50 values that are at least the same and preferablybetter, at least two times, preferably three times, more preferably fourtimes, even more preferably 5 times, 7 times or more than 7 times bettercompared to the IC50 value obtained for the reference IgG as defined bySEQ ID NO's: 1 and 2 or the reference Fab as defined by SEQ ID NO's: 3and 4 (see Example 1). In this plasma potency assay, the amino acidsequences of the invention may have IC50 values that are at least thesame and preferably better, at least two times, preferably three times,more preferably four times, even more preferably 5 times, 7 times ormore than 7 times better compared to the IC50 value obtained forTocilizumab (MRA).

In an assay for defining binding to membrane IL-6R on CHO cells, theamino acid sequences of the invention may have IC50 values between 10 nMand 100 pM, preferably between 5 nM and 100 pM, more preferably between2 nM and 10 pM or less, such as 2 nM or less.

In a preferred aspect, the amino acid sequences of the invention maycomprises two or more stretches of amino acid residues chosen from thefollowing:

-   -   a) SEQ ID NO's: 80-82; or    -   b) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with one of SEQ        ID NO's: 80-82, provided that the amino acid sequence comprising        said stretch of amino acid residues binds IL-6R with the same,        about the same, or a higher affinity compared to the amino acid        sequence comprising said stretch of amino acid residues without        the 2 or 1 amino acid difference, said affinity as measured by        surface plasmon resonance;    -   and/or    -   c) SEQ ID NO's: 84-91; or    -   d) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with one of SEQ        ID NO's: 84-91, provided that the amino acid sequence comprising        said stretch of amino acid residues binds IL-6R with the same,        about the same, or a higher affinity compared to the amino acid        sequence comprising said stretch of amino acid residues without        the 2 or 1 amino acid difference, said affinity as measured by        surface plasmon resonance;    -   and/or    -   e) SEQ ID NO's: 93-95; or    -   f) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with one of SEQ        ID NO's: 93-95, provided that the amino acid sequence comprising        said stretch of amino acid residues binds IL-6R with the same,        about the same, or a higher affinity compared to the amino acid        sequence comprising said stretch of amino acid residues without        the 2 or 1 amino acid difference, said affinity as measured by        surface plasmon resonance.    -   such that (i) when the first stretch of amino acid residues        corresponds to one of the amino acid sequences according to a),        or b), the second stretch of amino acid residues corresponds to        one of the amino acid sequences according to c), d), e) or        f); (ii) when the first stretch of amino acid residues        corresponds to one of the amino acid sequences according to c)        or d), the second stretch of amino acid residues corresponds to        one of the amino acid sequences according to a), b), e) or f);        or (iii) when the first stretch of amino acid residues        corresponds to one of the amino acid sequences according to e)        or f), the second stretch of amino acid residues corresponds to        one of the amino acid sequences according to a), b), c) or d).

Even more preferably, the amino acid sequences of the invention comprisethree or more stretches of amino acid residues, in which the firststretch of amino acid residues is chosen from the group consisting of;

-   -   a) SEQ ID NO's: 80-82; or    -   b) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with one of SEQ        ID NO's: 80-82, provided that the amino acid sequence comprising        said stretch of amino acid residues binds IL-6R with the same,        about the same, or a higher affinity compared to the amino acid        sequence comprising said stretch of amino acid residues without        the 2 or 1 amino acid difference, said affinity as measured by        surface plasmon resonance;    -   the second stretch of amino acid residues is chosen from the        group consisting of:    -   c) SEQ ID NO's: 84-91; or    -   d) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with one of SEQ        ID NO's: 84-91, provided that the amino acid sequence comprising        said stretch of amino acid residues binds IL-6R with the same,        about the same, or a higher affinity compared to the amino acid        sequence comprising said stretch of amino acid residues without        the 2 or 1 amino acid difference, said affinity as measured by        surface plasmon resonance;    -   and the third stretch of amino acid residues is chosen from the        group consisting of:    -   e) SEQ ID NO's: 93-95; or    -   f) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with one of SEQ        ID NO's: 93-95, provided that the amino acid sequence comprising        said stretch of amino acid residues binds IL-6R with the same,        about the same, or a higher affinity compared to the amino acid        sequence comprising said stretch of amino acid residues without        the 2 or 1 amino acid difference, said affinity as measured by        surface plasmon resonance.

It should be noted that the invention is not limited as to the origin ofthe amino acid sequence of the invention (or of the nucleotide sequenceof the invention used to express it), nor as to the way that the aminoacid sequence or nucleotide sequence of the invention is (or has been)generated or obtained. Thus, the amino acid sequences of the inventionmay be naturally occurring amino acid sequences (from any suitablespecies) or synthetic or semi-synthetic amino acid sequences.

In one specific, but non-limiting aspect, the amino acid sequence of theinvention may be an amino acid sequence that comprises an immunoglobulinfold or an amino acid sequence that, under suitable conditions (such asphysiological conditions) is capable of forming an immunoglobulin fold(i.e. by folding). Reference is inter alia made to the review by Halabyet al. (1999, J. Protein Eng. 12: 563-71). Preferably, when properlyfolded so as to form an immunoglobulin fold, the stretches of amino acidresidues may be capable of properly forming the antigen binding site forbinding the specific epitope on IL-6R; and more preferably capable ofbinding to their epitope on IL-6R with an affinity (suitably measuredand/or expressed as a K_(D)-value (actual or apparent), a K_(A)-value(actual or apparent), a k_(on)-rate and/or a k_(off)-rate, oralternatively as an IC₅₀ value, as further described herein) that is asdefined herein.

In another specific, but non-limiting aspect, the amino acid sequencesof the invention are immunoglobulin sequences. In particular, butwithout limitation, the amino acid sequences of the invention may beamino acid sequences that essentially consist of 4 framework regions(FR1 to FR4 respectively) and 3 complementarity determining regions(CDR1 to CDR3 respectively); or any suitable fragment of such an aminoacid sequence that still binds the specific epitope on IL-6R.

In such an amino acid sequence of the invention, the framework sequencesmay be any suitable framework sequences, and examples of suitableframework sequences will be clear to the skilled person, for example onthe basis the standard handbooks and the further disclosure and priorart mentioned herein.

The framework sequences are preferably (a suitable combination of)immunoglobulin framework sequences or framework sequences that have beenderived from immunoglobulin framework sequences (for example, bysequence optimization such as humanization or camelization). Forexample, the framework sequences may be framework sequences derived froma light chain variable domain (e.g. a V_(L)-sequence) and/or from aheavy chain variable domain (e.g. a V_(H)-sequence). When the amino acidsequence of the invention is a heavy chain variable domain sequence, itmay be a heavy chain variable domain sequence that is derived from aconventional four-chain antibody (such as, without limitation, a V_(H)sequence that is derived from a human antibody) or be a so-calledV_(HH)-sequence (as defined herein) that is derived from a so-called“heavy chain antibody” (as defined herein). In one particularlypreferred aspect, the framework sequences are either framework sequencesthat have been derived from a V_(HH)-sequence (in which said frameworksequences may optionally have been partially or fully humanized) or areconventional V_(H) sequences that have been camelized (as definedherein).

For a general description of heavy chain antibodies and the variabledomains thereof, reference is inter alia made to the prior art citedherein, as well as to the prior art mentioned on page 59 of WO08/020,079 and to the list of references mentioned on pages 41-43 of theInternational application WO 06/040153, which prior art and referencesare incorporated herein by reference.

The amino acid sequence of the invention may in particular be a domainantibody (or an amino acid sequence that is suitable for use as a domainantibody), a single domain antibody (or an amino acid sequence that issuitable for use as a single domain antibody), a “dAb” (or an amino acidsequence that is suitable for use as a dAb) or a Nanobody (as definedherein, and including but not limited to a V_(HH) sequence); othersingle variable domains, or any suitable fragment of any one thereof.

In particular, the amino acid sequence of the invention may be aNanobody® (as defined herein) or a suitable fragment thereof. [Note:Nanobody®, Nanobodies® and Nanocione® are registered trademarks ofAblynx N.V.] Such Nanobodies directed against IL-6R will also bereferred to herein as “Nanobodies of the invention”.

In general, a Nanobody can be defined as an amino acid sequence with the(general) structure

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

-   -   in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which one or more of the Hallmark residues are as defined in WO        08/020,079 (Tables A-3 to A-8).

Generally, Nanobodies (in particular V_(HH) sequences and partiallyhumanized Nanobodies) can in particular be characterized by the presenceof one or more “Hallmark residues” in one or more of the frameworksequences (as e.g. further described in WO 08/020,079, page 61, line 24to page 98, line 3).

In this respect, the amino acid sequences of the invention mayessentially consist of 4 framework regions (FR1 to FR4, respectively)and 3 complementarity determining regions (CDR1 to CDR3, respectively),in which:

-   -   CDR1 is chosen from the group consisting of:    -   a) SEQ ID NO's: 80-82; or    -   b) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with one of SEQ        ID NO's: 80-82, provided that the amino acid sequence comprising        said stretch of amino acid residues binds IL-6R with the same,        about the same, or a higher affinity compared to the amino acid        sequence comprising said stretch of amino acid residues without        the 2 or 1 amino acid difference, said affinity as measured by        surface plasmon resonance;    -   and/or    -   CDR2 is chosen from the group consisting of:    -   c) SEQ ID NO's: 84-91; or    -   d) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with one of SEQ        ID NO's: 84-91, provided that the amino acid sequence comprising        said stretch of amino acid residues binds IL-6R with the same,        about the same, or a higher affinity compared to the amino acid        sequence comprising said stretch of amino acid residues without        the 2 or 1 amino acid difference, said affinity as measured by        surface plasmon resonance;    -   and/or    -   CDR3 is chosen from the group consisting of:    -   e) SEQ ID NO's: 93-95; or    -   f) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with one of SEQ        ID NO's: 93-95, provided that the amino acid sequence comprising        said stretch of amino acid residues binds IL-6R with the same,        about the same, or a higher affinity compared to the amino acid        sequence comprising said stretch of amino acid residues without        the 2 or 1 amino acid difference, said affinity as measured by        surface plasmon resonance.

These preferred complementarity determining regions (CDR1 to CDR3,respectively) are also referred to as “CDR(s) of the invention”.

Preferably, the amino acid sequences of the invention essentiallyconsist of 4 framework regions (FR1 to FR4, respectively) and 3complementarity determining regions (CDR1 to CDR3, respectively), inwhich:

-   -   CDR1 is chosen from the group consisting of:    -   a) SEQ ID NO's: 80-82; or    -   b) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with one of SEQ        ID NO's: 80-82, provided that the amino acid sequence comprising        said stretch of amino acid residues binds IL-6R with the same,        about the same, or a higher affinity compared to the amino acid        sequence comprising said stretch of amino acid residues without        the 2 or 1 amino acid difference, said affinity as measured by        surface plasmon resonance;    -   and    -   CDR2 is chosen from the group consisting of:    -   c) SEQ ID NO's: 84-91; or    -   d) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with one of SEQ        ID NO's: 84-91, provided that the amino acid sequence comprising        said stretch of amino acid residues binds IL-6R with the same,        about the same, or a higher affinity compared to the amino acid        sequence comprising said stretch of amino acid residues without        the 2 or 1 amino acid difference, said affinity as measured by        surface plasmon resonance;    -   and    -   CDR3 is chosen from the group consisting of:    -   e) SEQ ID NO's: 93-95; or    -   f) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with one of SEQ        ID NO's: 93-95, provided that the amino acid sequence comprising        said stretch of amino acid residues binds IL-6R with the same,        about the same, or a higher affinity compared to the amino acid        sequence comprising said stretch of amino acid residues without        the 2 or 1 amino acid difference, said affinity as measured by        surface plasmon resonance.

Such Nanobodies may be derived in any suitable manner and from anysuitable source, and may for example be naturally occurring V_(HH)sequences (i.e. from a suitable species of Camelid) or synthetic orsemi-synthetic amino acid sequences.

In a specific aspect, the amino acid sequences or Nanobody of theinvention comprises at least SEQ ID NO: 80; or a stretch of amino acidresidues that has no more than 2, preferably no more than 1 amino aciddifference with SEQ ID NO: 80, provided that the amino acid sequence orNanobody comprising said stretch of amino acid residues binds IL-6R withthe same, about the same, or a higher affinity compared to the aminoacid sequence or Nanobody comprising said stretch of amino acid residueswithout the 2 or 1 amino acid difference, said affinity as measured bysurface plasmon resonance.

In another specific aspect, the amino acid sequence or Nanobody of theinvention comprises at least a stretch of amino acid residues chosenfrom SEQ ID NO's: 84, 89 or 91; or a stretch of amino acid residues thathas no more than 2, preferably no more than 1 amino acid difference withone of SEQ ID NO's: 84, 89 or 91, provided that the amino acid sequenceor Nanobody comprising said stretch of amino acid residues binds IL-6Rwith the same, about the same, or a higher affinity compared to theamino acid sequence or Nanobody comprising said stretch of amino acidresidues without the 2 or 1 amino acid difference, said affinity asmeasured by surface plasmon resonance.

In yet another specific aspect, the amino acid sequence or Nanobody ofthe invention comprises at least SEQ ID NO: 84; or a stretch of aminoacid residues that has no more than 2, preferably no more than 1 aminoacid difference with SEQ ID NO: 84, provided that the amino acidsequence or Nanobody comprising said stretch of amino acid residuesbinds IL-6R with the same, about the same, or a higher affinity comparedto the amino acid sequence or Nanobody comprising said stretch of aminoacid residues without the 2 or 1 amino acid difference, said affinity asmeasured by surface plasmon resonance.

In yet another specific aspect, the amino acid sequence or Nanobody ofthe invention comprises at least a stretch of amino acid residues chosenfrom SEQ ID NO's: 93-94; or a stretch of amino acid residues that has nomore than 2, preferably no more than 1 amino acid difference with one ofSEQ ID NO's: 93-94, provided that the amino acid sequence or Nanobodycomprising said stretch of amino acid residues binds IL-6R with thesame, about the same, or a higher affinity compared to the amino acidsequence or Nanobody comprising said stretch of amino acid residueswithout the 2 or 1 amino acid difference, said affinity as measured bysurface plasmon resonance.

In yet another specific aspect, the amino acid sequence or Nanobody ofthe invention comprises at least SEQ ID NO: 93; or a stretch of aminoacid residues that has no more than 2, preferably no more than 1 aminoacid difference with SEQ ID NO: 93, provided that the amino acidsequence or Nanobody comprising said stretch of amino acid residuesbinds IL-6R with the same, about the same, or a higher affinity comparedto the amino acid sequence or Nanobody comprising said stretch of aminoacid residues without the 2 or 1 amino acid difference, said affinity asmeasured by surface plasmon resonance.

In yet another specific aspect, the amino acid sequence or Nanobody ofthe invention comprises at least SEQ ID NO: 80 and HQ ID NO: 84.

In yet another specific aspect, the amino acid sequence or Nanobody ofthe invention comprises at least SEQ ID NO: 80 and SEQ ID NO: 93.

In yet another specific aspect, the amino acid sequence or Nanobody ofthe invention comprises at least SEQ ID NO: 84 and SEQ ID NO: 93.

In yet another specific aspect, the amino acid sequence or Nanobody ofthe invention comprises at least SEQ ID NO: 80, SEQ ID NO: 84 and SEQ IDNO: 93.

Other preferred combinations of CDR1, CDR2, and CDR3 sequences are alsoshown in Table A-1.

Preferred amino acid sequences of the invention may be selected from thegroup consisting of SEQ ID NO's: 60-69; a sequence that has no more than2, preferably no more than 1 amino acid difference in one, two or all ofits CDRs with one of SEQ ID NO's: 60-69, provided that the amino acidsequence with no more than 2, preferably no more than 1 amino aciddifference in one, two or all of its CDRs binds IL-6R with the same,about the same, or a higher affinity compared to the binding by the oneof SEQ ID NO's: 60-69, said affinity as measured by surface plasmonresonance; and a sequence that has no more than 2, preferably no morethan 1 amino acid difference with one of SEQ ID NO's: 60-69, providedthat the amino acid sequence with no more than 2, preferably no morethan 1 amino acid difference with one of SEQ ID NO's: 60-69 binds IL-6Rwith the same, about the same, or a higher affinity compared to thebinding by the one of SEQ ID NO's: 60-69, said affinity as measured bysurface plasmon resonance.

Such amino acid sequences of the invention should preferably be capableof specifically binding to the specific epitope on IL-6R, and even morepreferably capable of binding to the specific epitope on IL-6R with anaffinity (suitably measured and/or expressed as a K_(D)-value (actual orapparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or ak_(off)-rate, or alternatively as an IC₅₀ value, as further describedherein) that is as defined herein. Such amino acid sequences of theinvention should preferably also have a cell based potency and a plasmapotency as defined herein.

The amino acid sequences and Nanobodies provided by the invention arepreferably in essentially isolated form (as defined herein), or formpart of a protein or polypeptide of the invention (also referred to as“polypeptide of the invention” or “protein of the invention”), which maycomprise or essentially consist of one or more amino acid sequences orNanobodies of the invention and which may optionally further compriseone or more further amino acid sequences or Nanobodies (all optionallylinked via one or more suitable linkers).

Accordingly, in another aspect, the invention relates to a compound orconstruct, and in particular a protein or polypeptide (also referred toherein as a “compound of the invention” or “polypeptide of theinvention”, respectively) that comprises or essentially consists of oneor more amino acid sequences or Nanobodies of the invention (or suitablefragments thereof), and optionally further comprises one or more othergroups, residues, moieties or binding units. As will become clear to theskilled person from the further disclosure herein, such further groups,residues, moieties, binding units or amino acid sequences may or may notprovide further functionality to the amino acid sequence of theinvention (and/or to the compound, construct or polypeptide in which itis present) and may or may not modify the properties of the amino acidsequence or Nanobody of the invention.

For example, such further groups, residues, moieties or binding unitsmay be one or more additional amino acid sequences, such that thecompound, construct or polypeptide is a (fusion) protein or (fusion)polypeptide. In a preferred but non-limiting aspect, said one or moreother groups, residues, moieties or binding units are immunoglobulinsequences. Even more preferably, said one or more other groups,residues, moieties or binding units are chosen from the group consistingof domain antibodies, amino acid sequences that are suitable for use asa domain antibody, single domain antibodies, amino acid sequences thatare suitable for use as a single domain antibody, “dAb”'s, amino acidsequences that are suitable for use as a dAb, or Nanobodies.

Alternatively, such groups, residues, moieties or binding units may forexample be chemical groups, residues, moieties, which may or may not bythemselves be biologically and/or pharmacologically active. For example,and without limitation, such groups may be linked to the one or moreamino acid sequences or Nanobodies of the invention so as to provide a“derivative” of an amino acid sequence or polypeptide of the invention,as further described herein.

Also within the scope of the present invention are compounds, constructsor polypeptides, that comprise or essentially consist of one or morederivates as described herein, and optionally further comprise one ormore other groups, residues, moieties or binding units, optionallylinked via one or more linkers. Preferably, said one or more othergroups, residues, moieties or binding units are amino acid sequences.

In the compounds, constructs or polypeptides described above, the one ormore amino acid sequences or Nanobodies of the invention and the one ormore groups, residues, moieties or binding units may be linked directlyto each other and/or via one or more suitable linkers or spacers. Forexample, when the one or more groups, residues, moieties or bindingunits are amino acid sequences, the linkers may also be amino acidsequences, so that the resulting compound, construct or polypeptide is afusion (protein) or fusion (polypeptide).

The process of designing/selecting and/or preparing a compound orpolypeptide of the invention, starting from an amino acid sequence orNanobody of the invention, is also referred to herein as “formatting”said amino acid sequence or Nanobody of the invention; and an amino acidsequence or Nanobody of the invention that is made part of a compound orpolypeptide of the invention is said to be “formatted” or to be “in theformat of” said compound or polypeptide of the invention. Examples ofways in which an amino acid sequence or Nanobody of the invention can beformatted and examples of such formats will be clear to the skilledperson based on the disclosure herein; and such formatted amino acidsequences or Nanobodies form a further aspect of the invention.

For example, and without limitation, the one or more amino acidsequences or Nanobodies of the invention may be used as a binding unitin such a protein or polypeptide, which may optionally contain one ormore further amino acid sequences that can serve as a binding unit (i.e.against another epitope on IL-6R and/or against one or more otherantigens, proteins or targets than IL-6R), so as to provide amonovalent, multivalent, multiparatopic or multispecific polypeptide ofthe invention, respectively, all as described herein. The presentinvention thus also relates to a compound, construct or polypeptidewhich is a monovalent construct comprising or essentially consisting ofan amino acid sequence or Nanobody of the invention. The presentinvention thus also relates to a compound, construct or polypeptidewhich is a multivalent construct, such as e.g. a bivalent or trivalentconstruct. The present invention also relates to a compound, constructor polypeptide which is a multispecific construct, such as e.g. abispecific or trispecific construct. The present invention also relatesto a compound, construct or polypeptide which is a multiparatopicconstruct, such as e.g. a bisparatopic or triparatopic construct.

In one specific aspect of the invention, a compound of the invention ora polypeptide of the invention may have an increased half-life, comparedto the corresponding amino acid sequence or Nanobody of the invention.Some preferred, but non-limiting examples of such compounds andpolypeptides will become clear to the skilled person based on thefurther disclosure herein, and for example comprise amino add sequences,Nanobodies or polypeptides of the invention that have been chemicallymodified to increase the half-life thereof (for example, by means ofpegylation); amino acid sequences or Nanobodies of the invention thatcomprise at least one additional binding site for binding to a serumprotein (such as serum albumin); or polypeptides of the invention thatcomprise at least one amino acid sequence or Nanobody of the inventionthat is linked to at least one moiety (and in particular at least oneamino acid sequence) that increases the half-life of the amino acidsequence or Nanobody of the invention. Examples of polypeptides, aminoacid sequences or Nanobodies of the invention that comprise suchhalf-life extending moieties will become clear to the skilled personbased on the further disclosure herein; and for example include, withoutlimitation, polypeptides in which the one or more amino acid sequencesor Nanobodies of the invention are suitable linked to one or more serumproteins or fragments thereof (such as (human) serum albumin or suitablefragments thereof) or to one or more binding units that can bind toserum proteins (such as, for example, domain antibodies, amino acidsequences that are suitable for use as a domain antibody, single domainantibodies, amino acid sequences that are suitable for use as a singledomain antibody, “dAb”'s, amino acid sequences that are suitable for useas a dAb, or Nanobodies can bind to serum proteins such as serum albumin(such as human serum albumin), serum immunoglobulins such as IgG, ortransferrine; reference is made to the further description andreferences mentioned herein); polypeptides in which an amino acidsequence or Nanobody of the invention is linked to an Fc portion (suchas a human Fc) or a suitable part or fragment thereof; or polypeptidesin which the one or more amino acid sequences or Nanobodies of theinvention are suitable linked to one or more small proteins or peptidesthat can bind to serum proteins (such as, without limitation, theproteins and peptides described in WO 91/01743, WO 01/45746, WO02/076489).

Generally, the compounds or polypeptides of the invention with increasedhalf-life preferably have a half-life that is at least 1.5 times,preferably at least 2 times, such as at least 5 times, for example atleast 10 times or more than 20 times, greater than the half-life of thecorresponding amino acid sequence or Nanobody of the invention per se.

In a preferred, but non-limiting aspect, such compounds or polypeptidesof the invention have a serum half-life that is increased with more than1 hour, preferably more than 2 hours, more preferably more than 6 hours,such as more than 12 hours, or even more than 24, 48 or 72 hours,compared to the corresponding amino acid sequence or Nanobody of theinvention per se.

In another preferred, but non-limiting aspect, such compounds orpolypeptides of the invention exhibit a serum half-life in human of atleast about 12 hours, preferably at least 24 hours, more preferably atleast 48 hours, even more preferably at least 72 hours or more. Forexample, compounds or polypeptides of the invention may have a half-lifeof at least 5 days (such as about 5 to 10 days), preferably at least 9days (such as about 9 to 14 days), more preferably at least about 10days (such as about 10 to 15 days), or at least about 11 days (such asabout 11 to 16 days), more preferably at least about 12 days (such asabout 12 to 18 days or more), or more than 14 days (such as about 14 to19 days).

Such a protein, polypeptide, compound or construct may also be inessentially isolated form (as defined herein).

Some preferred compounds, constructs or polypeptides of the inventioninclude the following polypeptide sequences:

-   -   a) SEQ ID NO's: 70-72;    -   b) a polypeptide sequence that has no more than 2, preferably no        more than 1 amino acid difference in one, two or all of its CDRs        of the invention with one of SEQ ID NO's: 70-72, provided that        the polypeptide sequence with no more than 2, preferably no more        than 1 amino acid difference in one, two or all of its CDRs of        the invention binds IL-6R with the same, about the same, or a        higher affinity compared to the binding by the one of SEQ ID        NO's: 70-72, said affinity as measured by surface plasmon        resonance;    -   c) a polypeptide sequence that has no more than 2, preferably no        more than 1 amino acid difference with one of SEQ ID NO's:        70-72, provided that the polypeptide sequence with no more than        2, preferably no more than 1 amino acid difference with one of        SEQ ID NO's: 70-72 binds IL-6R with the same, about the same, or        a higher affinity compared to the binding by the one of SEQ ID        NO's: 70-72, said affinity as measured by surface plasmon        resonance.

Polypeptides with these sequences show advantageous properties for useas pharmacologically active agents such as e.g. good bindingcharacteristics (high affinity and/or avidity), high efficacy and/orpotency, in addition to their capacity to (partially or totally) blockthe IL-6/IL-6R interaction and/or inhibit signalization through, IL-6,IL-6R and/or the IL-6/IL-6R complex.

More in particular, these polypeptides and compounds of the inventioncan bind to IL-6R with an affinity (suitably measured and/or expressedas a K_(D)-value (actual or apparent), a K_(A)-value (actual orapparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively as anIC₅₀ value, as further described herein) preferably such that they:

-   -   bind to hIL-6R with a dissociation constant (K_(D)) of 1 nM to 1        pM moles/litre or less, preferably 500 pM to 1 pM moles/litre or        less, more preferably 100 pM to 1 pM moles/litre or less, or        even more preferably about 50 pM to 1 pM or less;        and/or such that they:    -   bind to cyno IL-6R with a dissociation constant (K_(D)) of 1 nM        to 1 pM moles/litre or less, preferably 500 pM to 1 pM        moles/litre or less, more preferably 100 pM to 1 pM moles/litre        or less, or even more preferably about 50 pM to 1 pM or less;        and/or such that they:    -   bind to hIL-6R with a k_(on)-rate of between 10⁴ M⁻¹s⁻¹ about        10⁷ M⁻¹s⁻¹, preferably between 10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more        preferably about 10⁶ M⁻¹s⁻¹ or more;        and/or such that they:    -   bind to cyno IL-6R with a k_(on)-rate of between 10⁴ M⁻¹s⁻¹ to        about 10⁷ M⁻¹s⁻¹, preferably between 10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹,        more preferably about 10⁶ M⁻¹s⁻¹ or more;        and/or such that they:    -   bind to hIL-6R with a k_(off) rate between 10⁻³ s⁻¹        (t_(1/2)=0.69 s) and 10⁻⁶ s⁻¹ (providing a near irreversible        complex with a t_(1/2) of multiple days), preferably between        10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹, more preferably between 10⁻⁵ s⁻¹ and        10⁻⁶s⁻¹, such as about 10⁻⁵ s⁻¹ or lower;        and/or such that they:    -   bind to cyno IL-6R with a k_(off) rate between 10⁻³ s⁻¹        (t_(1/2)=0.69 s) and 10⁻⁶ s⁻¹ (providing a near irreversible        complex with a t_(1/2) of multiple days), preferably between        10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹, more preferably between 10⁻⁵ s⁻¹ and 10⁻⁶        s⁻¹, such as about 10⁻⁵ s⁻¹ or lower.

Some preferred IC50 values for binding of the polypeptides and compoundsof the invention to IL-6R will become clear from the further descriptionand examples herein.

For example, in the TF-1 assay as described by Kitamura et al. (J. CellPhysiol. 1989; 140: 323), the polypeptides and compounds of theinvention may have IC50 values (at 100 IU/mL IL-6) between 10 nM and 50pM, preferably between 5 nM and 50 pM, more preferably between 1 nM and50 pM or less, such as about 750 or 500 pM or less. In this TF-1 assaythe polypeptides and compounds of the invention may have IC50 values (at5000 IU/mL IL-6) between 50 nM and 1 nM, preferably between 25 nM and 1nM, more preferably between 10 nM and 1 nM or less, such as about 8 nMor less. In this TF-1 assay, the polypeptides and compounds of theinvention may have IC50 values that are at least the same and preferablybetter, at least two times, preferably three times, more preferably fourtimes, even more preferably 5 times, 7 times or more than 7 times bettercompared to the IC50 value obtained for the reference IgG as defined bySEQ ID NO's: 1 and 2 or the reference Fab as defined by SEQ ID NO's: 3and 4 (see Example 1). In this TF-1 assay, the amino acid sequences ofthe invention may have IC50 values that are at least the same andpreferably better, at least two times, preferably three times, morepreferably four times, even more preferably 5 times, 7 times or morethan 7 times better compared to the IC₅₀ value obtained for Tocilizumab(MRA).

in a plasma potency assay at ECSO values of IL-6 (e.g. in the presenceof 27.29 ng/mL IL-6 as described in Example 45) the polypeptides andcompounds of the invention may have IC50 values between 500 pM and 50pM, preferably between 250 pM and 50 pM, more preferably between 200 pMand 50 pM or less, such as 150 pM or less. In a plasma potency assay atEC95 values of IL-6 (e.g. in the presence of 885 ng/mL IL-6 as describedin Example 45) the polypeptides and compounds of the invention may haveIC50 values between 1000 pM and 100 pM, preferably between 750 pM and100 pM, more preferably between 500 pM and 100 pM or less, such as 400pM or less. In this plasma potency assay, the polypeptides and compoundsof the invention may have IC50 values that are at least the same andpreferably better, at least two times, preferably three times, morepreferably four times, even more preferably 5 times, 7 times or morethan 7 times better compared to the IC50 value obtained for thereference IgG as defined by SEQ ID NO's: 1 and 2 or the reference Fab asdefined by SEQ ID NO's: 3 and 4 (see Example 1). In this plasma potencyassay, the amino acid sequences of the invention may have IC50 valuesthat are at least the same and preferably better, at least two times,preferably three times, more preferably four times, even more preferably5 times, 7 times or more than 7 times better compared to the IC50 valueobtained for Tocilizumab (MRA).

In an assay for defining binding to membrane IL-6R on CHO cells, thepolypeptides and compounds of the invention may have IC50 values between10 nM and 100 pM, preferably between 5 nM and 100 pM, more preferablybetween 2 nM and 10 pM or less, such as 2 nM or less.

In another specific aspect, the polypeptide, compound or construct ofthe invention essentially consists of the amino acid sequence of SEQ IDNO: 70.

In another specific aspect, the polypeptide, compound or construct ofthe invention essentially consists of the amino acid sequence of SEQ IDNO: 71

The compounds or polypeptides of the invention can generally be preparedby a method which comprises at least the step of suitably linking theone or more amino acid sequence, Nanobody or monovalent construct of theinvention to the one or more further groups, residues, moieties orbinding units, optionally via the one or more suitable linkers, so as toprovide the compound or polypeptide of the invention. Polypeptides ofthe invention can also be prepared by a method which generally comprisesat least the steps of providing a nucleic acid that encodes apolypeptide of the invention, expressing said nucleic acid in a suitablemanner, and recovering the expressed polypeptide of the invention. Suchmethods can be performed in a manner known per se, which will be clearto the skilled person, for example on the basis of the methods andtechniques further described herein.

Accordingly, the present invention also relates to the use of an aminoacid sequence, a Nanobody or a monovalent construct of the invention inpreparing a multivalent compound, construct or polypeptide. The methodfor the preparation of a multivalent compound, construct or polypeptidewill comprise the linking of an amino acid sequence, a Nanobody or amonovalent construct of the invention to at least one other group,residue, moiety or binding unit, optionally via one or more linkers.

Generally, when an amino acid sequence or Nanobody of the invention (ora compound, construct or polypeptide comprising the same) is intendedfor administration to a subject (for example for therapeutic and/ordiagnostic purposes as described herein), it is preferably either anamino acid sequence or Nanobody that does not occur naturally in saidsubject; or, when it does occur naturally in said subject, inessentially isolated form (as defined herein).

The amino acid sequences, Nanobodies, polypeptides and compounds of theinvention are directed against IL-6R from humans. However, they shouldpreferably also be cross-reactivity with IL-6R from cynomolgus monkeys(Macaca fascicularis), by which is meant that these amino acidsequences, Nanobodies, polypeptides and compounds are also “directedagainst” (as defined herein) and/or are capable of specific binding to(as defined herein) IL-6R from cynomolgus monkeys (Macaca fascicularis).Such cross-reactivity, may have advantages from a drug development pointof view, since it allows the amino acid sequences, Nanobodies,polypeptides and compounds against human IL-6R to be tested in acynomolgus monkey disease model.

An amino acid sequence or Nanobody of the invention (as well ascompounds, constructs and polypeptides comprising the same) is“cross-reactive” with IL-6R from humans and from cynomolgus monkey meansthat the amino acid sequence or Nanobody of the invention (as well ascompounds, constructs and polypeptides comprising the same) binds toIL-6R from a cynomolgus monkey with an affinity (suitably measuredand/or expressed as a K_(D)-value (actual or apparent), a K_(A)-value(actual or apparent), a k_(on)-rate and/or a k_(off)-rate, oralternatively as an IC₅₀ value, as further described herein) that is thesame or at least 70% of (preferably at least 80% of, more preferably atleast 90% of, even more preferably at least 95% of) the affinity withwhich said amino acid sequence or Nanobody of the invention (as well ascompounds, constructs and polypeptides comprising the same) binds toIL-6R from humans. For the IL-6R sequence and the corresponding cDNAsequence of cynomolgus monkey, reference is also made to WO 09/010,539filed by Ablynx N.V. on Jul. 16, 2008 entitled “Receptor forinterleukin-6 (IL-6) from Macaca fascicularis”; see SEQ ID NO: 3 andFIG. 1B for the cDNA sequence and SEQ ID NO: 4 and FIG. 3B for the aminoacid sequence).

It is also within the scope of the invention to use parts, fragments,analogs, mutants, variants, alleles and/or derivatives of the amino acidsequences and polypeptides of the invention, and/or to use proteins orpolypeptides comprising or essentially consisting of one or more of suchparts, fragments, analogs, mutants, variants, alleles and/orderivatives, as long as these are suitable for the uses envisagedherein. Such parts, fragments, analogs, mutants, variants, allelesand/or derivatives will usually contain (at least part of) a functionalantigen-binding site for binding against the specific epitope on IL-6R;and more preferably will be capable of specific binding to the specificepitope on IL-6R, and even more preferably capable of binding to thespecific epitope on IL-6R with an affinity (suitably measured and/orexpressed as a K_(D)-value (actual or apparent), a K_(A)-value (actualor apparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively asan IC₅₀ value, as further described herein) that is as defined herein.Such parts, fragments, analogs, mutants, variants, alleles and/orderivatives will usually also have a cell based potency and a plasmapotency as defined herein. Some non-limiting examples of such parts,fragments, analogs, mutants, variants, alleles, derivatives, proteinsand/or polypeptides will become clear from the further descriptionherein. Additional fragments or polypeptides of the invention may alsobe provided by suitably combining (i.e. by linking or genetic fusion)one or more (smaller) parts or fragments as described herein.

In another aspect, the invention also relates to a nucleic acid or anucleotide sequence that encodes an amino acid sequence of theinvention, a Nanobody of the invention or a polypeptide of the invention(or a suitable fragment thereof). Such a nucleic acid will also bereferred to herein as a “nucleic acid of the invention” and may forexample be in the form of a genetic construct, as further describedherein. Accordingly, the present invention also relates to a nucleicacid or nucleotide sequence that is in the form of a genetic construct.

The nucleotide sequences of the invention may be naturally occurringnucleotide sequences or synthetic or semi-synthetic sequences, and mayfor example be sequences that are isolated by PCR from a suitablenaturally occurring template (e.g. DNA or RNA isolated from a cell),nucleotide sequences that have been isolated from a library (and inparticular, an expression library), nucleotide sequences that have beenprepared by introducing mutations into a naturally occurring nucleotidesequence (using any suitable technique known per se, such as mismatchPCR), nucleotide sequence that have been prepared by PCR usingoverlapping primers, or nucleotide sequences that have been preparedusing techniques for DNA synthesis known per se.

In another aspect, the invention relates to a host or host cell thatexpresses (or that under suitable circumstances is capable ofexpressing) an amino acid sequence of the invention, a Nanobody of theinvention and/or a polypeptide of the invention; and/or that contains anucleic acid of the invention. Some preferred but non-limiting examplesof such hosts or host cells will become clear from the furtherdescription herein.

The invention further relates to a product or composition containing orcomprising at least one amino acid sequence of the invention (or asuitable fragment thereof), at least one Nanobody of the invention, atleast one polypeptide of the invention, at least one compound orconstruct of the invention, at least one monovalent construct of theinvention and/or at least one nucleic acid of the invention, andoptionally one or more further components of such compositions known perse, i.e. depending on the intended use of the composition. Such aproduct or composition may for example be a pharmaceutical composition(as described herein), a veterinary composition or a product orcomposition for diagnostic use (as also described herein). Somepreferred but non-limiting examples of such products or compositionswill become clear from the further description herein.

The invention further relates to methods for preparing the amino acidsequences, Nanobodies, polypeptides, nucleic acids, host cells, productsand compositions described herein. The method for producing an aminoacid sequence of the invention, a Nanobody of the invention, apolypeptide of the invention, or a monovalent construct of the inventionmay comprise the following steps:

-   -   a) expressing, in a suitable host cell or host organism or in        another suitable expression system, a nucleic acid or nucleotide        sequence of the invention, or a genetic construct of the        invention;    -   optionally followed by:    -   b) isolating and/or purifying the amino acid sequence, the        Nanobody, the polypeptide, or the monovalent construct of the        invention thus obtained.        The method for producing an amino acid sequence, a Nanobody, a        polypeptide, or a monovalent construct of the invention may        comprise the steps of:    -   a) cultivating and/or maintaining a host or host cell of the        invention under conditions that are such that said host or host        cell expresses and/or produces at least one amino acid sequence,        Nanobody, polypeptide, or a monovalent construct of the        invention,    -   optionally followed by:    -   b) isolating and/or purifying the amino acid sequence, the        Nanobody, the polypeptide, or the monovalent construct of the        invention thus obtained.

The invention further relates to applications and uses of the amino acidsequences, polypeptides, compounds, constructs, nucleic acids, hostcells, products and compositions described herein, as well as to methodsfor the prevention and/or treatment for diseases and disordersassociated with IL-6R. Some preferred but non-limiting applications anduses will become clear from the further description herein.

The amino acid sequences, Nanobodies, polypeptides, compounds,constructs and compositions of the present invention can generally beused to modulate, and in particular inhibit and/or prevent, binding ofIL-6R to IL-6 and subsequent binding of the IL-6/IL-6R complex to gp130and thus to modulate, and in particular inhibit and/or prevent, thesignalling that is mediated by IL-6R, IL-6, IL-6/IL-6R complex and/orgp130, to modulate the biological pathways in which IL-6R, IL-6, theIL-6/IL-6R complex and/or gp130 are involved, and/or to modulate thebiological mechanisms, responses and effects associated with suchsignalling or these pathways.

In one aspect, the invention provides amino acid sequences, Nanobodies,polypeptides, constructs and compounds that are, and/or that can be usedas, an antagonist of IL-6R, of IL-6R-mediated signalling, and/or of thebiological pathways mechanisms, responses and/or effects in which IL-6Rand/or IL-6R mediated signalling are involved.

In this respect, the amino acid sequences, Nanobodies, polypeptides,compounds, constructs and compositions of the present invention are suchthat they (a) specifically bind (as defined herein) to the IL-6receptor; and (b) are capable of downregulating the IL-6 receptor and/orare capable of inhibiting, decreasing or downregulating the signallingof the IL-6 receptor and/or the pathway(s), mechanism(s) or signallingin which IL-6 or IL-6R is involved. As will be clear to the skilledperson, such an amino acid sequence, Nanobody, polypeptide, compound orconstruct can generally be used as an antagonist of IL-6, of the IL-6receptor and/or of the biological pathways, mechanisms or effects inwhich IL-6, IL-6R and/or IL-6/IL-6R complex mediated signalling isinvolved. Any such decrease or downregulation (which can be at least 1%,such as at least 5%, as at least 10%, or more than 10%, or up to 50% or100% or more in a relevant parameter, compared to the same parameterunder conditions in which the amino acid sequence, Nanobody,polypeptide, compound or construct is not bound to the IL-6 receptor),may be measured in any suitable manner known per se, for example usingone of the assays used described above and/or in the Experimental Partand/or mentioned herein.

More in particular, and in addition to (a) and (b) above, suchantagonistic amino acid sequences, Nanobodies, polypeptides, compoundsand constructs bind to IL-6R in such a way that (c) binding of IL-6 toIL-6R is blocked, inhibited or reduced; compared to the binding of IL-6to its receptor without the presence of the amino acid sequence,Nanobody or polypeptide of the invention.

Without limitation, such antagonistic amino acid sequences, Nanobodies,polypeptides, compounds and constructs may bind to a specific epitope onIL-6R close to the IL-6 interaction side on IL-6R.

Also, in addition to (a) and (b) above, and in addition to (c) above,such antagonistic amino acid sequences and polypeptides may bind toIL-6R (i.e. as such or as present in the IL-6/IL-6R complex) in such away that (d) the formation of the IL-6/IL-6R complex is inhibited oraffected (e.g. fully or partially disrupted) in such a way that thebinding of the complex to—e.g. its affinity for—gp130 is reduced (orreversely, that the binding of gp 130 to—e.g. its affinity for—thecomplex is reduced), so that the signaling induced/mediated by thebinding of the complex to gp130 is modulated (e.g. reduced); compared tothe formation of the complex and its binding to gp130 without thepresence of the amino acid sequence, Nanobody, polypeptide, compound orconstruct of the invention.

The amino acid sequences, Nanobodies, polypeptides, compounds,constructs and compositions of the invention are also preferably (butwithout limitation) such that they effect a decrease (i.e. by at least 1percent such as by at least 10 percent, preferably by at least 30percent, more preferably by at least 50 percent, even more preferably byat least 75 percent or more) or a total inhibition of the induction ofC-reactive protein (CRP) in a mammal (such as in a human subject or in asuitable animal model for inflammation such as the a cynomolgus monkey)when they are administered to said mammal in a therapeutically relevantamount compared to a mammal not receiving the amino acid sequence,Nanobody, polypeptide, compound, construct or compositions of theinvention.

The amino acid sequences, Nanobodies, polypeptides, compounds,constructs and compositions of the invention are also preferably (butwithout limitation) such that they effect a decrease (i.e. by at least 1percent such as by at least 10 percent, preferably by at least 30percent, more preferably by at least 50 percent, even more preferably byat least 75 percent or more) or a total inhibition of the induction ofthe platelet count in a mammal (such as in a human subject or in asuitable animal model for inflammation such as the a cynomolgus monkey)when they are administered to said mammal in a therapeutically relevantamount compared to a mammal not receiving the amino acid sequence,Nanobody, polypeptide, compound, construct or compositions of theinvention.

The amino acid sequences, Nanobodies, polypeptides, compounds,constructs and compositions of the invention are also preferably (butwithout limitation) such that they effect a decrease (i.e. by at least 1percent such as by at least 10 percent, preferably by at least 30percent, more preferably by at least 50 percent, even more preferably byat least 75 percent or more) or a total inhibition of the induction offibrinogen in a mammal (such as in a human subject or in a suitableanimal model for inflammation such as the cynomolgus monkey) when theyare administered to said mammal in a therapeutically relevant amountcompared to a mammal not receiving the amino acid sequence, Nanobody,polypeptide, compound, construct or compositions of the invention.

As such, the amino acid sequences, polypeptides, compounds, constructsand compositions of the present invention can be used for the preventionand/or treatment of diseases and disorders associated with IL-6R, withIL-6, with the IL-6/IL-6R complex (optionally in further complex withgp130), and/or with the signaling pathway(s) and/or the biologicalfunctions and responses in which IL-6, IL-6R and/or the IL-6/IL-6Rcomplex (optionally in further complex with gp130) are involved, and inparticular for the prevention and/or treatment of diseases and disordersassociated with IL-6R, IL-6, with the IL-6/IL-6R complex (optionally infurther complex with gp130), and/or with the signaling pathway(s) and/orthe biological functions and responses in which IL-6R, IL-6 and/or theIL-6/IL-6R complex (optionally in further complex with gp130) areinvolved, which are characterized by excessive and/or unwantedsignalling mediated by IL-6R or by the pathway(s) in which IL-6R isinvolved. Examples of such diseases and disorders associated with IL-6R,with IL-6, with the IL-6/IL-6R complex, and/or with the signalingpathway(s) and/or the biological functions and responses in which IL-6,IL-6R and/or the IL-6/IL-6R complex are involved, will be clear to theskilled person based on the disclosure herein, and for example includethe following diseases and disorders: sepsis (Starnes et al., 1999, J.Immunol., 148: 1968) and various forms of cancer such as multiplemyeloma disease (MM), renal cell carcinoma (RCC), plasma cell leukaemia(Klein et al., 1991, Blood, 78: 1198-1204), lymphoma,B-lymphoproliferative disorder (BLPD) and prostate cancer. Non-limitingexamples of other diseases caused by excessive IL-6 production orsignalling include bone resorption (osteoporosis) (Roodman et al., 1992,J. Clin. Invest. 89: 45-52; Jilka et al., 1992, Science, 257: 88-91),cachexia (Strassman et al., 1992, J. Clin. Invest., 89: 1681-1684),psoriasis, mesangial proliferative glomerulonephritis, Kaposi's sarcoma,AIDS-related lymphoma (Emilie et al., 1994, Blood, 84: 2472-2479),inflammatory diseases and disorder such as rheumatoid arthritis,systemic onset juvenile idiopathic arthritis, hypergammaglobulinemia(Grau et al., 1990, J. Exp. Med., 172: 1505-1508); Crohn's disease,ulcerative colitis, systemic lupus erythematosus (SLE), multiplesclerosis, Castleman's disease, IgM gammopathy, cardiac myxoma, asthma(in particular allergic asthma) and autoimmune insulin-dependentdiabetes mellitus (Campbell et al., 1991, J. Clin. Invest. 87: 739-742).Other IL-6R, IL-6 and/or IL-6/IL-6R complex related disorders will beclear to the skilled person. Such diseases and disorders are alsogenerally referred to herein as “IL-6R related diseases and disorders”.

Thus, without being limited thereto, the amino acid sequences,Nanobodies, polypeptides, compounds, constructs and compositions of theinvention can for example be used to prevent and/or to treat alldiseases and disorders that are currently being prevented or treatedwith active principles that can modulate IL-6R-mediated signalling, suchas those mentioned in the prior art cited above. It is also envisagedthat the amino acid sequences, Nanobodies, polypeptides, compounds,constructs and compositions of the invention can be used to preventand/or to treat all diseases and disorders for which treatment with suchactive principles is currently being developed, has been proposed, orwill be proposed or developed in future. In addition, it is envisagedthat, because of their favourable properties as further describedherein, the amino acid sequences, Nanobodies, polypeptides, compounds,constructs and compositions of the present invention may be used for theprevention and treatment of other diseases and disorders than those forwhich these known active principles are being used or will be proposedor developed; and/or that the amino acid sequences, Nanobodies,polypeptides, compounds, constructs and compositions of the presentinvention may provide new methods and regimens for treating the diseasesand disorders described herein.

Accordingly, the present invention also relates to a method for theprevention and/or treatment of at least one disease or disorder that canbe prevented and/or treated by administering, to a subject in needthereof, an amino acid sequence of the invention, a Nanobody of theinvention, a polypeptide of the invention, or a monovalent construct ofthe invention, said method comprising administering, to a subject inneed thereof, a pharmaceutically active amount of at least one aminoacid sequence of the invention, Nanobody of the invention, polypeptideof the invention, compound of the invention, or (monovalent) constructof the invention, or a composition of the invention.

The invention also relates to the use of an amino acid sequence of theinvention, a Nanobody of the invention, a polypeptide of the invention,compound of the invention, or (monovalent) construct of the invention inthe preparation of a pharmaceutical composition for prevention and/ortreatment of at least one of the diseases and disorders associated withIL-6, with IL-6R, with the IL-6/IL-6R complex and/or with the signallingpathways and/or the biological functions and responses in which IL-6,IL-6R and/or the IL-6/IL-6R complex are involved; and/or for use in oneor more of the methods described herein.

The invention further relates to an amino acid sequence of theinvention, a Nanobody of the invention, a polypeptide of the invention,compound of the invention, or (monovalent) construct of the inventionfor use in the prevention and/or treatment of at least one of thediseases and disorders associated with IL-6, with IL-6R, with theIL-6/IL-6R complex and/or with the signalling pathways and/or thebiological functions and responses in which IL-6, IL-6R and/or theIL-6/IL-6R complex are involved; and/or for use in one or more of themethods described herein.

In particular, the present invention provides amino acid sequences,Nanobodies, proteins, polypeptides, compounds and/or constructs that aresuitable for prophylactic, therapeutic and/or diagnostic use in awarm-blooded animal, and in particular in a mammal, and more inparticular in a human being.

More in particular, the present invention provides such amino acidsequences, Nanobodies, proteins, polypeptides, compounds and/orconstructs that can be used for the prevention, treatment, alleviationand/or diagnosis of one or more IL-6R related disorders (as definedherein) in a warm-blooded animal, in particular in a mammal, and more inparticular in a human being.

Other applications and uses of the amino acid sequences, Nanobodies,polypeptides and compounds of the invention will become clear to theskilled person from the further disclosure herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Analysis of the immune response in llamas 81 and 82 by ELISA asdescribed in Example 2.

FIG. 2: Analysis of the immune response in llamas 81 and 82 by FACS asdescribed in Example 2. Legend: Ll81 pre: pre-immune serum from llama81; Ll81 PBL1: serum collected at day 28 from llama 81; Ll82 pre:pre-immune serum from llama 82; Ll82 PBL2: serum collected at day 43from llama 82.

FIG. 3: Schematic representation of the Alphascreen assays used toidentify Nanobodies against the IL-6 binding site on IL-6R.

FIG. 4: Amino acid sequences of anti-IL-6R Nanobodies.

FIG. 5: SDS-PAGE of purified Nanobodies obtained as described in Example6.

FIG. 6: Inhibition of the IL-6/IL-6R interaction by selected Nanobodiesas measured in alphascreen. MAb BR-6 and the reference Fab fragment(described in Example 1) were used as a control.

FIG. 7: Binding of the anti-IL-6R Nanobodies to U266 cells analyzed byFACS.

FIG. 8: Binding of the anti-IL-6R Nanobodies to U266 cells in theabsence (top) and presence (bottom) of human plasma.

FIG. 9: Individual observed (symbols) and model predicted (solid line)plasma concentration-time profiles of IL6R304 in cynomolgus monkey afteri.v. administration with 1 mg/kg (∘), 5 mg/kg (Δ), 10 mg/kg (+), 25mg/kg (x) and 100 mg/kg (⋄).

FIG. 10: Binding of the Nanobodies to mouse and human IL-6R. In eachgroup of three bars, the bar on the left indicates human IL 6R, themiddle bar indicates mouse IL 6R and the bar on the right indicates theblank.

FIG. 11: SDS-PAGE of purified bispecific Nanobodies.

FIG. 12: Inhibition of IL-6/IL-6R interaction by bispecific Nanobodiesas measured in alphascreen.

FIG. 13: FACS analysis of bivalent Nanobodies binding to U266 cells.

FIG. 14: FACS analysis of trivalent Nanobodies binding to U266 cells.

FIG. 15: Alignment of IL6R03, IL6R04 and IL6R13 sequences with 5 mosthomologous human germlines. For further explanation see Example 23.

FIGS. 16A and B: Amino acid sequences of sequence optimized variants ofIL6R03, IL6R04 and IL6R13. For further explanation see Example 23.

FIG. 17: Inhibition of the IL-6/IL-6R interaction by sequence optimizedvariants of IL6R03.

FIG. 18: Inhibition of the IL-6/IL-6R interaction by sequence optimizedvariants of IL6R04.

FIG. 19: Inhibition of the IL-6/IL-6R interaction by sequence optimizedvariants of IL6R13.

FIG. 20: Inhibition of the IL-6/IL-6R interaction by sequence optimizedvariants of IL6R13.

FIG. 21: Inhibition of the IL-6/IL-6R interaction by wild-type andsequence optimized anti-IL-6R Nanobodies.

FIG. 22: Cell-based potency of sequence optimized Nanobodies versuswildtype Nanobodies.

FIG. 23: Plasma potency ELISA of sequence optimized Nanobodies in human(A) and cyno (B) plasma.

FIG. 24: Plasma potency ELISA of sequence optimized Nanobodies at theEC50 (left) and the EC95 (right) of IL-6.

FIG. 25: Epitope mapping of IL-6R Nanobodies as described in Example 29:competition assay on an IL-6R-coated (A-C) or an IL-6-coated chip.

FIG. 26: Amino acid sequences of affinity matured IL6R65 variants.

FIG. 27: Binding curves of Nanobody IL6R65 (referred to as parentNanobody) and its affinity matured variants.

FIG. 28: Evaluation of IL6R65 and 5 affinity matured variants in a humanand cyno plasma potency assay.

FIG. 29: Inhibition of IL-6-dependent proliferation of TF-1 cells byaffinity matured Nanobodies. Cells were grown in the presence of 2 ng/mlhuman IL-6 and various concentrations of Nanobody. Proliferation wasmeasured by 3H-thymidine incorporation.

FIG. 30: Competition of IL-6 with two affinity matured Nanobodies (B-C)in comparison with the competition of IL-6 with the IL6R65 (A) asmeasured in Biacore.

FIG. 31: Sequences of IL-6R binding Nanobodies after 2nd round ofaffinity maturation (combinatorial libraries CDR1/2+CDR3).

FIG. 32: Inhibition of IL-6-dependent proliferation of TF-1 cells. Cellswere grown in the presence of 2 ng/ml human IL-6 and variousconcentrations of Nanobody. Proliferation was measured by 3H-thymidineincorporation.

FIG. 33: Evaluation of IL6R65 and 2nd round affinity matured variants ina human and cyno plasma potency assay. Parent=IL6R65.

FIG. 34: Binding of IL6R65 and PMP20A11 to human PBMCs in full blood.

FIG. 35: inhibition of membrane IL-6R activity by formatted Nanobodiesand reference IgG. TF-1 cells were pre-incubated with a dilution seriesof IL6R304 (▪), IL6R305 (▴), IL6R306 (▾) or reference IgG (•) afterwhich proliferation was induced with 10011.1/mL IL-6. After 72 hincubation, cell proliferation was assessed by incorporation of ³Hthymidine. Mean±s.e. of triplicate measurements is shown.

FIG. 36: Inhibition of membrane IL-6R by the formatted Nanobodies andthe reference IgG at high levels of IL-6. TF-1 cells were pre-incubatedwith a dilution series of 20A11 (♦), IL6R304 (▪), IL6R305 (▴), IL6R306(▾) or the reference IgG (•) after which proliferation was induced with5000 IU/mL IL-6. After 72 h incubation, cell proliferation was assessedby incorporation of ³H thymidine. Mean±s.e. of triplicate measurementsis shown.

FIG. 37: Effect of IL6R304 and IL6R305 on proliferation of TF-1 cells.TF-1 cells were seeded at a density of 12500 cells/well and incubatedwith or without 50 nM IL6R304 or IL6R305. Proliferation was induced with100 IU/mL IL-6 or cells were incubated in the absence of growth factors.After 72 h incubation, cell proliferation was assessed by incorporationof ³H thymidine. Each data point was measured 30 times. Mean±s.e. isshown.

FIG. 38: Plasma potency ELISA in human plasma. Neutralization of bindingof human IL-6 to plasma sIL-6R by the reference IgG (•), IL6R20A11 (♦),IL6R304 (▪), IL6R305 (▴), IL6R306 (▾) or an irrelevant NB (x). Themean±s.e. of duplicate measurements is shown. A, B: competition with 25ng/mL of IL-6 (EC50). C,D: competition with 885 ng/mL of IL-6 (EC95).

FIG. 39: Binding of IL6R20A11 and formatted variants to CHO cells. CHOcells expressing IL-6R (A) or negative CHO cells (B) were incubated withIL6R20A11 (♦), IL6R304 (▪), IL6R305 (▴) or IL6R306 (▾). Bound Nanobodywas detected using MAb c1.5.3.1 and anti-mouse-PE.

FIG. 40: Binding of IL-6R Nanobodies on human PBL in full blood. Left:lymphocytes (L, black), monocytes (M, dark gray) and granulocytes (G,light gray) were gated based on FSC/SSC properties. Middle: backgroundPE fluorescence of the three gated populations. Right: PE fluorescenceafter incubation with 1 μM of IL6R305.

FIG. 41: Binding of IL-6R Nanobodies to human PBL. EDTA-treated bloodfrom 2 donors was incubated with IL6R20A11 (♦), IL6R304 (▪), IL6R305 (▴)or IL6R306 (▾). Bound Nanobody was detected using MAb cl.5.3.1 andanti-mouse-PE. A: lymphocytes; B: monocytes; C: granulocytes.

FIG. 42: Binding curves of formatted affinity matured Nanobodies onhuman and cyno serum albumin.

FIG. 43: Binding curves of formatted affinity matured Nanobodies tohuman and cyno IL-6R.

FIG. 44: Plasma potency ELISA in cyno plasma. Neutralization of bindingof human IL-6 to cyno plasma sIL-6R by the reference IgG (•), IL6R20A11(♦), IL6R304 (▪), IL6R305 (▴), IL6R306 (▾) or an irrelevant NB (x).

FIG. 45: Cross-reactivity of IL6R20A11 with sIL-6R from other species.Left: binding of IL6R20A11 to human sIL-6R on the plate afterpre-incubation with recombinant sIL-6R from human (•), cyno (▪) or mouse(▴). Right: binding of IL6R20A11 to human sIL-61R after pre-incubationwith human (•), cyno (▪), mouse (▴) or guinea pig (▾) plasma.

FIG. 46: Competitive binding ELISA. IL6R20A11 (0.05 nM) waspre-incubated with different concentrations of IL-6R (•), LIF-R (▪),CNTF-R (▴), OSM-R (▾) or IL-11R/Fc (♦). Free IL6R20A11 was captured onsIL-6R and detected via anti-His.

FIG. 47: Study design for in vivo PK/PD analysis of IL6R304 and IL6R305.

FIG. 48: Effect of the reference IgG, IL6R304 and IL6R305 on CRP levelsincreased by hiL-6 in individual cynomolgus monkeys. (A) Animals 27, 28and 29 served as negative controls and received only hIL-6. Thereference IgG (B, closed black symbols), different doses of IL6R304 (C,blue symbols) and different doses of IL6R305 (ID, red symbols) were i.v.administered followed by s.c. injections of hIL-6 at a dose of 5 μg/kgonce a day for 7 days.

FIG. 49: Mean CRP levels for all groups obtained in the in vivo PK/PDstudy with IL6R304 and 1 L6R305.

FIG. 50: Effect of the reference IgG, IL6R304 and IL6R305 on fibrinogenlevels increased by hIL-6 in individual cynomolgus monkeys. (A) Animals27, 28 and 29 served as negative controls and received only hIL-6.Reference IgG (B, closed black symbols), different doses of IL6R304 (C,blue symbols) and different doses of IL6R305 (D, red symbols) were i.v.administered followed by s.c. injections of hIL-6 at a dose of 5 mg/kgonce a day for 7 days. Results were normalized to the basal levels.

FIG. 51: Mean fibrinogen levels for all groups obtained in the in vivoPK/PD study with IL6R304 and IL6R305.

FIG. 52: Effect of the reference IgG, IL6R304 and IL6R305 on plateletcounts increased by hIL-6 in individual cynomolgus monkeys. (A) Animals27, 28 and 29 served as negative controls and received only hIL-6.Reference IgG (A, closed black symbols), different doses of IL6R304 (B,blue symbols) and different doses of IL6R305 (C, red symbols) were i.v.administered followed by s.c. injections of hIL-6 at a dose of 5 μg/kgonce a day for 7 days. Results were normalized to the basal levels.

FIG. 53: Mean platelet counts for all groups obtained in the in vivoPK/PD study with IL6R304 and IL6R305.

FIG. 54: Individual observed plasma concentration-time plots after i.v.bolus administration of IL6R304 (0.4-2-10 mg/kg) in cynomolgus monkeys.11m, 12m and 13f are the three graphs on the left hand side; 17m, 18fand 14m are the middle graphs and 15m and 16f are the graphs on theright hand side.

FIG. 55: Individual observed plasma concentration-time plots after i.v.bolus administration of IL6R305 (0.4-2-10 mg/kg) in cynomolgus monkeys.19m, 20f and 21f are the three graphs on the left hand side; 25m, 26fand 22m are the middle graphs and 23m and 24f are the graphs on theright hand side.

FIG. 56: Mean observed plasma concentration vs. time plots after i.v.bolus administration of IL6R304 (0.4-2-10 mg/kg) and IL6R202 (2 mg/kg,dose-normalized to 0.4-10 mg/kg) in cynomolgus monkeys.

FIG. 57: Immunodetection of anti-IL6R304 antibodies at pre-dose anddifferent days post i.v. administration of IL6R304. ELISA plates werecoated with IL6R304. The legend in each of the figures corresponds tothe bar graph groupings starting from left to right.

FIG. 58: Immunodetection of anti-IL6R304 antibodies at pre-dose anddifferent days post i.v. administration of 116R304. ELISA plates werecoated with IL6R300. The legend in each of the figures corresponds tothe bar graph groupings starting from left to right.

FIG. 59: Immunodetection of anti-IL6R304 antibodies at pre-dose anddifferent days post i.v. administration of 116R304. ELISA plates werecoated with ALB8. The legend in each of the figures corresponds to thebar graph groupings starting from left to right.

FIG. 60: Immunodetection of anti-116R305 antibodies at pre-dose anddifferent days post i.v. administration of IL6R305. ELISA plates werecoated with IL6R305. The legend in each of the figures corresponds tothe bar graph groupings starting from left to right.

FIG. 61: Immunodetection of anti-IL6R305 antibodies at pre-dose anddifferent days post i.v. administration of IL6R305. ELISA plates werecoated with IL6R300. The legend in each of the figures corresponds tothe bar graph groupings starting from left to right.

FIG. 62: Immunodetection of anti-IL6R305 antibodies at pre-dose anddifferent days post i.v. administration of IL6R305. ELISA plates werecoated with ALB8. The legend in each of the figures corresponds to thebar graph groupings starting from left to right.

FIG. 63: Plasma sIL-6R levels in cynomolgus monkeys after a single IVbolus dose of IL6R Nanobodies. A: 2 animals treated with Ref IgG at 5mg/kg (▪, ) or vehicle (▴), B: 3 animals with IL6R304 at 0.04 mg/kg, C:3 animals with IL6R305 at 0.04 mg/kg.

FIG. 64: Total plasma sIL-6R levels in cynomolgus monkeys after a singleIV bolus dose of IL6R Nanobodies. A: animals treated with IL6R304 (▴, ▪,), Ref IgG (▾) or vehicle (⋄); B: animals treated with IL6R305 (▴, ▪,), Ref IgG (▾) or vehicle (⋄). The mean±s.e. per group is shown.

FIG. 65: Total plasma IL-6 levels in cynomolgus monkeys after a singleIV bolus dose of IL6R Nanobodies. Total plasma IL-6 concentrations, bothendogenous cyno and injected human IL-6, were measured via the Gyrolabplatform. Samples that were below the limit of quantification aredepicted as 9.6 pg/mL. A: IL6R304; B: IL6R305; C: positive (Ref IgG) andnegative (buffer) control. The mean per treatment group±s.e. (n=3 for0.4 and 2 mg/kg; n=2 for 10 mg/kg) is depicted. D: endogenous cynoplasma IL-6 concentrations in individual animals after IV bolusinjection of 10 mg/kg IL6R304 (▪, ), IL6R305 (▴, ▾) or irrelevantNanobody (⋄, •). The mean±s.e. of 2 measurements is shown.

FIG. 66: Total (A) and free (B) sIL6R plasma levels in cynomolgus monkeyafter a single i.v. administration of different doses of IL6R304. Meanplasma concentrations of either biomarker±SD per group is shown. Vehicleor IL6R304 at given doses were administered at time point 0. For thelegend, see FIG. 66B.

FIG. 67: Total and free sIL6R plasma levels and IL6R304 concentration incynomolgus monkey after a single i.v. administration of 25 mg/kgIL6R304. The mean±SD for the specific dose group is shown. Total sIL6R(solid line, square symbols), free sIL6R (solid line, round symbols) andIL6R304 concentrations (dotted line, delta) are plotted versus time(days).

FIG. 68: Individual observed (symbols) and model-predicted (solid lines)total sIL6R concentration-time plots after i.v, administration with 1mg/kg (∘), 5 mg/kg (Δ), 10 mg/kg (+), 25 mg/kg (x) and 100 mg/kg (⋄) ofIL6R304.

FIG. 69: Mean plasma concentration-time profiles of IL6R304 incynomolgus monkey after i.v. bolus injection with 1, 5, 10, 25 or 100mg/kg of IL6R304.

FIG. 70: Open three-compartmental pharmacokinetic model with linear andnon-linear clearance from the central compartment. CL_(NON-IL6R) is thelinear non-IL6R mediated clearance, V_(c) the volume of the centralcompartment, V_(d) the volume of the deep peripheral compartment, CL_(d)the inter-compartmental flow between central and deep compartment, V_(s)the volume of the shallow peripheral compartment, CL_(s) theinter-compartmental flow between central and shallow compartment andCL_(IL6R) is the non-linear IL6R-mediated clearance (with V_(max) themaximum metabolic rate and K_(m) the IL6R304 concentration correspondingto 50% of V_(max)).

DETAILED DESCRIPTION

In the present description, examples and claims:

-   a) Unless indicated or defined otherwise, all terms used have their    usual meaning in the art, which will be clear to the skilled person.    Reference is for example made to the standard handbooks, such as    Sambrook at al., “Molecular Cloning: A Laboratory Manual” (2^(nd)    Ed.), Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); F.    Ausubel et al., eds., “Current protocols in molecular biology”,    Green Publishing and Wiley Interscience, New York (1987); Lewin,    “Genes II”, John Wiley & Sons, New York, N.Y., (1985); Old et al.,    “Principles of Gene Manipulation: An Introduction to Genetic    Engineering”, 2nd edition, University of California Press, Berkeley,    Calif. (1981); Roitt at al., “Immunology” (6th. Ed.),    Mosby/Elsevier, Edinburgh (2001); Roitt et al., Roitt's Essential    Immunology, 10^(th) Ed. Blackwell Publishing, UK (2001); and Janeway    at al., “Immunobiology” (6th Ed.), Garland Science    Publishing/Churchill Livingstone, New York (2005), as well as to the    general background art cited herein;-   b) Unless indicated otherwise, the term “immunoglobulin    sequence”—whether used herein to refer to a heavy chain antibody or    to a conventional 4-chain antibody—is used as a general term to    include both the full-size antibody, the individual chains thereof,    as well as all parts, domains or fragments thereof (including but    not limited to antigen-binding domains or fragments such as V_(HH)    domains or V_(H)/V_(L) domains, respectively). In addition, the term    “sequence” as used herein (for example in terms like “immunoglobulin    sequence”, “antibody sequence”, “variable domain sequence”, “V_(HH)    sequence” or “protein sequence”), should generally be understood to    include both the relevant amino acid sequence as well as nucleic    acids or nucleotide sequences encoding the same, unless the context    requires a more limited interpretation. Also, the term “nucleotide    sequence” as used herein also encompasses a nucleic acid molecule    with said nucleotide sequence, so that the terms “nucleotide    sequence” and “nucleic acid” should be considered equivalent and are    used interchangeably herein;-   c) Unless indicated otherwise, all methods, steps, techniques and    manipulations that are not specifically described in detail can be    performed and have been performed in a manner known per se, as will    be clear to the skilled person. Reference is for example again made    to the standard handbooks and the general background art mentioned    herein and to the further references cited therein; as well as to    for example the following reviews Presta, 2006, Adv. Drug Deliv.    Rev., 58 (5-6): 640-56; Levin and Weiss, 2006, Mol. Biosyst., 2(1):    49-57; Irving et al., 2001, J. Immunol. Methods, 248 (1-2): 31-45;    Schmitz et al., 2000, Placenta, 21 Suppl. A, S106-12; Gonzales et    al., 2005, Tumour Biol., 26(1): 31-43, which describe techniques for    protein engineering, such as affinity maturation and other    techniques for improving the specificity and other desired    properties of proteins such as immunoglobulins.-   d) Amino acid residues will be indicated according to the standard    three-letter or one-letter amino acid code, as mentioned in Table    A-2;

TABLE A-2 one-letter and three-letter amino acid code Nonpolar, AlanineAla A uncharged Valine Val V (at pH 6.0-7.0)⁽³⁾ Leucine Leu L IsoleucineIle I Phenylalanine Phe F Methionine⁽¹⁾ Met M Tryptophan Trp W ProlinePro P Polar, uncharged Glycine⁽²⁾ Gly G (at pH 6.0-7.0) Serine Ser SThreonine Thr T Cysteine Cys C Asparagine Asn N Glutamine Gln Q TyrosineTyr Y Polar, Lysine Lys K charged Arginine Arg R (at pH 6.0-7.0)Histidine⁽⁴⁾ His H Aspartate Asp D Glutamate Glu E Notes: ⁽¹⁾Sometimesalso considered to be a polar uncharged amino acid. ⁽²⁾Sometimes alsoconsidered to be a nonpolar uncharged amino acid. ⁽³⁾As will be clear tothe skilled person, the fact that an amino acid residue is referred toin this Table as being either charged or uncharged at pH 6.0 to 7.0 doesnot reflect in any way on the charge said amino acid residue may have ata pH lower than 6.0 and/or at a pH higher than 7.0; the amino acidresidues mentioned in the Table can be either charged and/or unchargedat such a higher or lower pH, as will be clear to the skilled person.⁽⁴⁾As is known in the art, the charge of a His residue is greatlydependant upon even small shifts in pH, but a His residue can generallybe considered essentially uncharged at a pH of about 6.5.

-   e) For the purposes of comparing two or more nucleotide sequences,    the percentage of “sequence identity” between a first nucleotide    sequence and a second nucleotide sequence may be calculated by    dividing [the number of nucleotides in the first nucleotide sequence    that are identical to the nucleotides at the corresponding positions    in the second nucleotide sequence] by [the total number of    nucleotides in the first nucleotide sequence] and multiplying by    [100%], in which each deletion, insertion, substitution or addition    of a nucleotide in the second nucleotide sequence—compared to the    first nucleotide sequence—is considered as a difference at a single    nucleotide (position).    -   Alternatively, the degree of sequence identity between two or        more nucleotide sequences may be calculated using a known        computer algorithm for sequence alignment such as NCBI Blast        v2.0, using standard settings.    -   Some other techniques, computer algorithms and settings for        determining the degree of sequence identity are for example        described in WO 04/037999, EP 0967284, EP 1085089, WO 00/55318,        WO 00/78972, WO 98/49185 and GB 2357768-A.    -   Usually, for the purpose of determining the percentage of        “sequence identity” between two nucleotide sequences in        accordance with the calculation method outlined hereinabove, the        nucleotide sequence with the greatest number of nucleotides will        be taken as the “first” nucleotide sequence, and the other        nucleotide sequence will be taken as the “second” nucleotide        sequence;-   f) For the purposes of comparing two or more amino acid sequences,    the percentage of “sequence identity” between a first amino acid    sequence and a second amino acid sequence (also referred to herein    as “amino acid identity”) may be calculated by dividing [the number    of amino acid residues in the first amino acid sequence that are    identical to the amino acid residues at the corresponding positions    in the second amino acid sequence] by [the total number of amino    acid residues in the first amino acid sequence] and multiplying by    [100%], in which each deletion, insertion, substitution or addition    of an amino acid residue in the second amino acid sequence—compared    to the first amino acid sequence—is considered as a difference at a    single amino acid residue (position), i.e. as an “amino acid    difference” as defined herein.    -   Alternatively, the degree of sequence identity between two amino        acid sequences may be calculated using a known computer        algorithm, such as those mentioned above for determining the        degree of sequence identity for nucleotide sequences, again        using standard settings. Usually, for the purpose of determining        the percentage of “sequence identity” between two amino acid        sequences in accordance with the calculation method outlined        hereinabove, the amino acid sequence with the greatest number of        amino acid residues will be taken as the “first” amino acid        sequence, and the other amino acid sequence will be taken as the        “second” amino acid sequence.    -   Also, in determining the degree of sequence identity between two        amino acid sequences, the skilled person may take into account        so-called “conservative” amino acid substitutions, which can        generally be described as amino acid substitutions in which an        amino acid residue is replaced with another amino acid residue        of similar chemical structure and which has little or        essentially no influence on the function, activity or other        biological properties of the polypeptide. Such conservative        amino acid substitutions are well known in the art, for example        from WO 04/037999, GB 335 768-A, WO 98/49185, WO 00/46383 and WO        01/09300; and (preferred) types and/or combinations of such        substitutions may be selected on the basis of the pertinent        teachings from WO 04/037999 as well as WO 98/49185 and from the        further references cited therein.    -   Such conservative substitutions preferably are substitutions in        which one amino acid within the following groups (a)-(e) is        substituted by another amino acid residue within the same        group: (a) small aliphatic, nonpolar or slightly polar residues:        Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged        residues and their (uncharged) amides: Asp, Asn, Glu and        Gln; (c) polar, positively charged residues: His, Arg and        Lys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val        and Cys; and (e) aromatic residues: Phe, Tyr and Trp.    -   Particularly preferred conservative substitutions are as        follows: Ala into Gly or into Ser; Arg into Lys; Asn into Gln or        into His; Asp into Glu; Cys into Ser; Gln into Asn; Glu into        Asp; Gly into Ala or into Pro; His into Asn or into Gln; Ile        into Leu or into Val; Leu into Ile or into Val; Lys into Arg,        into Gln or into Glu; Met into Leu, into Tyr or into Ile; Phe        into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp        into Tyr; Tyr into Trp; and/or Phe into Val, into Ile or into        Leu. Any amino acid substitutions applied to the polypeptides        described herein may also be based on the analysis of the        frequencies of amino acid variations between homologous proteins        of different species developed by Schulz et al. (1978,        “Principles of Protein Structure”, Springer-Verlag), on the        analyses of structure forming potentials developed by Chou and        Fasman (1974, Biochemistry 13: 211, and 1978, Adv. Enzymol., 47:        45-149), and on the analysis of hydrophobicity patterns in        proteins developed by Eisenberg et at. (1984, Proc. Natl. Acad.        Sci. USA 81: 140-144), Kyte and Doolittle (1981, J. Molec. Biol.        157: 105-132), and Goldman et al. 1986, Ann. Rev. Biophys. Chem.        15: 321-353), all incorporated herein in their entirety by        reference. Information on the primary, secondary and tertiary        structure of Nanobodies is given in the description herein and        in the general background art cited above. Also, for this        purpose, the crystal structure of a V_(HH) domain from a llama        is for example given by Desmyter et at. (1996, Nature Structural        Biology, 3 (9): 803), Spinelli et al. (1996, Natural Structural        Biology, 3: 752-757) and Decanniere et at. (1999, Structure, 7        (4): 361). Further information about some of the amino acid        residues that in conventional V_(H) domains form the V_(H)/V_(L)        interface and potential camelizing substitutions on these        positions can be found in the prior art cited above.-   g) Amino acid sequences and nucleic acid sequences are said to be    “exactly the same” if they have 100% sequence identity (as defined    herein) over their entire length;-   h) When comparing two amino acid sequences, the term “amino acid    difference” refers to an insertion, deletion or substitution of a    single amino acid residue on a position of the first sequence,    compared to the second sequence; it being understood that two amino    acid sequences can contain one, two or more such amino acid    differences;-   i) When a nucleotide sequence or amino acid sequence is said to    “comprise” another nucleotide sequence or amino acid sequence,    respectively, or to “essentially consist of” another nucleotide    sequence or amino acid sequence, this may mean that the latter    nucleotide sequence or amino acid sequence has been incorporated    into the firstmentioned nucleotide sequence or amino acid sequence,    respectively, but more usually this generally means that the    firstmentioned nucleotide sequence or amino acid sequence comprises    within its sequence a stretch of nucleotides or amino acid residues,    respectively, that has the same nucleotide sequence or amino acid    sequence, respectively, as the latter sequence, irrespective of how    the firstmentioned sequence has actually been generated or obtained    (which may for example be by any suitable method described herein).    By means of a non-limiting example, when an amino acid sequence of    the invention is said to comprise a stretch of amino acid residues,    this may mean that said stretch of amino acid residues has been    incorporated into the amino acid sequence of the invention, but more    usually this generally means that the amino acid sequence of the    invention contains within its sequence the stretch of amino acid    residues irrespective of how said amino acid sequence of the    invention has been generated or obtained. When a Nanobody of the    invention is said to comprise a CDR sequence, this may mean that    said CDR sequence has been incorporated into the Nanobody of the    invention, but more usually this generally means that the Nanobody    of the invention contains within its sequence a stretch of amino    acid residues with the same amino acid sequence as said CDR    sequence, irrespective of how said Nanobody of the invention has    been generated or obtained. It should also be noted that when the    latter amino acid sequence has a specific biological or structural    function, it preferably has essentially the same, a similar or an    equivalent biological or structural function in the firstmentioned    amino acid sequence (in other words, the firstmentioned amino acid    sequence is preferably such that the latter sequence is capable of    performing essentially the same, a similar or an equivalent    biological or structural function). For example, when a Nanobody of    the invention is said to comprise a CDR sequence or framework    sequence, respectively, the CDR sequence and framework are    preferably capable, in said Nanobody, of functioning as a CDR    sequence or framework sequence, respectively. Also, when a    nucleotide sequence is said to comprise another nucleotide sequence,    the firstmentioned nucleotide sequence is preferably such that, when    it is expressed into an expression product (e.g. a polypeptide), the    amino acid sequence encoded by the latter nucleotide sequence forms    part of said expression product (in other words, that the latter    nucleotide sequence is in the same reading frame as the    firstmentioned, larger nucleotide sequence).-   j) A nucleic acid sequence or amino add sequence is considered to be    “(in) essentially isolated (form)”—for example, compared to its    native biological source and/or the reaction medium or cultivation    medium from which it has been obtained—when it has been separated    from at least one other component with which it is usually    associated in said source or medium, such as another nucleic acid,    another protein/polypeptide, another biological component or    macromolecule or at least one contaminant, impurity or minor    component. In particular, a nucleic acid sequence or amino acid    sequence is considered “essentially isolated” when it has been    purified at least 2-fold, in particular at least 10-fold, more in    particular at least 100-fold, and up to 1000-fold or more. A nucleic    acid sequence or amino acid sequence that is “in essentially    isolated form” is preferably essentially homogeneous, as determined    using a suitable technique, such as a suitable chromatographical    technique, such as polyacrylamide-gel electrophoresis;-   k) The term “domain” as used herein generally refers to a globular    region of an amino acid sequence (such as an antibody chain, and in    particular to a globular region of a heavy chain antibody), or to a    polypeptide that essentially consists of such a globular region.    Usually, such a domain will comprise peptide loops (for example 3 or    4 peptide loops) stabilized, for example, as a sheet or by disulfide    bonds. The term “binding domain” refers to such a domain that is    directed against an antigenic determinant (as defined herein);-   l) The term “antigenic determinant” refers to the epitope on the    antigen recognized by the antigen-binding molecule (such as an amino    acid sequence, a Nanobody or a polypeptide of the invention) and    more in particular by the antigen-binding site of said molecule. The    terms “antigenic determinant” and “epitope” may also be used    interchangeably herein.-   m) An amino acid sequence (such as a Nanobody, an antibody, a    polypeptide of the invention, or generally an antigen binding    protein or polypeptide or a fragment thereof) that can    (specifically) bind to, that has affinity for and/or that has    specificity for a specific antigenic determinant, epitope, antigen    or protein (or for at least one part, fragment or epitope thereof)    is said to be “against” or “directed against” said antigenic    determinant, epitope, antigen or protein.-   n) The term “specificity” refers to the number of different types of    antigens or antigenic determinants to which a particular    antigen-binding molecule or antigen-binding protein (such as an    amino acid sequence, a Nanobody or a polypeptide of the invention)    molecule can bind. The specificity of an antigen-binding protein can    be determined based on affinity and/or avidity. The affinity,    represented by the equilibrium constant for the dissociation of an    antigen with an antigen-binding protein (K_(D)), is a measure for    the binding strength between an antigenic determinant and an    antigen-binding site on the antigen-binding protein: the lesser the    value of the K_(D), the stronger the binding strength between an    antigenic determinant and the antigen-binding molecule    (alternatively, the affinity can also be expressed as the affinity    constant (K_(A)), which is 1/K_(D)). As will be clear to the skilled    person (for example on the basis of the further disclosure herein),    affinity can be determined in a manner known per se, depending on    the specific antigen of interest. Avidity is the measure of the    strength of binding between an antigen-binding molecule (such as an    amino acid sequence, a Nanobody or polypeptide of the invention) and    the pertinent antigen, Avidity is related to both the affinity    between an antigenic determinant and its antigen binding site on the    antigen-binding molecule and the number of pertinent binding sites    present on the antigen-binding molecule. Typically, antigen-binding    proteins will bind to their antigen with a dissociation constant    (K_(D)) of 10⁻⁵ to 10⁻¹² moles/liter or less, and preferably 10⁻⁷ to    10⁻¹² moles/liter or less and more preferably 10⁻⁸ to 10⁻¹²    moles/liter (i.e. with an association constant (K_(A)) of 10⁵ to    10¹² liter/moles or more, and preferably 10⁷ to 10¹² liter/moles or    more and more preferably 10⁸ to 10¹² liter/moles). Any H_(D) value    greater than 10⁴ mol/liter (or any K_(A) value lower than 10⁴ M⁻¹)    liters/mol is generally considered to indicate non-specific binding.    Preferably, a monovalent immunoglobulin sequence of the invention    will bind to the desired antigen with an affinity less than 500 nM,    preferably less than 200 nM, more preferably less than 10 nM, such    as less than 500 pM. Specific binding of an antigen-binding protein    to an antigen or antigenic determinant can be determined in any    suitable manner known per se, including, for example, Scatchard    analysis and/or competitive binding assays, such as    radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich    competition assays, and the different variants thereof known per se    in the art; as well as the other techniques mentioned herein.    -   The dissociation constant may be the actual or apparent        dissociation constant, as will be clear to the skilled person.        Methods for determining the dissociation constant will be clear        to the skilled person, and for example include the techniques        mentioned herein. In this respect, it will also be clear that it        may not be possible to measure dissociation constants of more        then 10⁻⁴ moles/liter or 10⁻³ moles/liter (e.g. of 10⁻²        moles/liter). Optionally, as will also be clear to the skilled        person, the (actual or apparent) dissociation constant may be        calculated on the basis of the (actual or apparent) association        constant (K_(A)), by means of the relationship [K_(D)=1/K_(A)].        The affinity denotes the strength or stability of a molecular        interaction. The affinity is commonly given as by the K_(D), or        dissociation constant, which has units of mol/liter (or M). The        affinity can also be expressed as an association constant,        K_(A), which equals 1/K_(D) and has units of (mol/liter)⁻¹ (or        M⁻¹). In the present specification, the stability of the        interaction between two molecules (such as an amino acid        sequence, Nanobody or polypeptide of the invention and its        intended target) will mainly be expressed in terms of the K_(D)        value of their interaction; it being clear to the skilled person        that in view of the relation K_(A)=1/K_(D), specifying the        strength of molecular interaction by its K_(D) value can also be        used to calculate the corresponding K_(A) value. The K_(D)-value        characterizes the strength of a molecular interaction also in a        thermodynamic sense as it is related to the free energy (DG) of        binding by the well known relation DG=RT.ln(K_(D)) (equivalently        DG=−RT.ln(K_(A))), where R equals the gas constant, T equals the        absolute temperature and ln denotes the natural logarithm.    -   The K_(D) for biological interactions which are considered        meaningful (e.g. specific) are typically in the range of 10⁻¹⁰M        (0.1 nM) to 10⁻⁵M (10000 nM). The stronger an interaction is,        the lower is its K_(D).    -   The K_(D) can also be expressed as the ratio of the dissociation        rate constant of a complex, denoted as k_(off), to the rate of        its association, denoted k_(on) (so that K_(D)=k_(off)/k_(on)        and K_(A)=k_(on)/k_(off)). The off-rate k_(off) has units s⁻¹        (where s is the SI unit notation of second). The on-rate k_(on)        has units M⁻¹s⁻¹. The on-rate may vary between 10² M⁻¹s⁻¹ to        about 10⁷ M⁻¹s⁻¹, approaching the diffusion-limited association        rate constant for bimolecular interactions. The off-rate is        related to the half-life of a given molecular interaction by the        relation t_(1/2)=ln(2)/k_(off). The off-rate may vary between        10⁻⁶ s⁻¹ (near irreversible complex with a t_(1/2) of multiple        days) to 1 s⁻¹ (t_(1/2)=0.69 s). The affinity of a molecular        interaction between two molecules can be measured via different        techniques known per se, such as the well known surface plasmon        resonance (SPR) biosensor technique (see for example Ober et        al., Intern. Immunology, 13, 1551-1559, 2001) where one molecule        is immobilized on the biosensor chip and the other molecule is        passed over the immobilized molecule under flow conditions        yielding k_(on), k_(off) measurements and hence K_(D) (or K_(A))        values. This can for example be performed using the well-known        BIACORE instruments. It will also be clear to the skilled person        that the measured K_(D) may correspond to the apparent K_(D) if        the measuring process somehow influences the intrinsic binding        affinity of the implied molecules for example by artefacts        related to the coating on the biosensor of one molecule. Also,        an apparent K_(D) may be measured if one molecule contains more        than one recognition sites for the other molecule. In such        situation the measured affinity may be affected by the avidity        of the interaction by the two molecules.    -   Another approach that may be used to assess affinity is the        2-step ELISA (Enzyme-Linked Immunosorbent Assay) procedure of        Friguet et al. (1985, J. Immunol. Methods, 77: 305-19). This        method establishes a solution phase binding equilibrium        measurement and avoids possible artefacts relating to adsorption        of one of the molecules on a support such as plastic. However,        the accurate measurement of K_(D) may be quite labor-intensive        and as consequence, often apparent K_(D) values are determined        to assess the binding strength of two molecules. It should be        noted that as long all measurements are made in a consistent way        (e.g. keeping the assay conditions unchanged) apparent K_(D)        measurements can be used as an approximation of the true K_(D)        and hence in the present document K_(D) and apparent K_(D)        should be treated with equal importance or relevance.    -   Finally, it should be noted that in many situations the        experienced scientist may judge it to be convenient to determine        the binding affinity relative to some reference molecule. For        example, to assess the binding strength between molecules A and        B, one may e.g. use a reference molecule C that is known to bind        to B and that is suitably labelled with a fluorophore or        chromophore group or other chemical moiety, such as biotin for        easy detection in an ELISA or FACS (Fluorescent activated cell        sorting) or other format (the fluorophore for fluorescence        detection, the chromophore for light absorption detection, the        biotin for streptavidin-mediated ELISA detection). Typically,        the reference molecule C is kept at a fixed concentration and        the concentration of A is varied for a given concentration or        amount of B. As a result an IC₅₀ value is obtained corresponding        to the concentration of A at which the signal measured for C in        absence of A is halved. Provided K_(D ref), the K_(D) of the        reference molecule, is known, as well as the total concentration        c_(ref) of the reference molecule, the apparent K_(D) for the        interaction A-B can be obtained from following formula:        K_(D)=IC₅₀/(1+c_(ref)/K_(D ref)). Note that if        c_(ref)<<K_(D ref), K_(D)≈IC₅₀. Provided the measurement of the        IC₅₀ is performed in a consistent way (e.g. keeping c_(ref)        fixed) for the binders that are compared, the strength or        stability of a molecular interaction can be assessed by the IC₅₀        and this measurement is judged as equivalent to K_(D) or to        apparent K_(D) throughout this text.-   o) The half-life of an amino acid sequence, Nanobody, compound or    polypeptide of the invention can generally be defined as the time    taken for the serum concentration of the amino acid sequence,    Nanobody, compound or polypeptide to be reduced by 50%, in vivo, for    example due to degradation of the sequence or compound and/or    clearance or sequestration of the sequence or compound by natural    mechanisms. The in vivo half-life of an amino acid sequence,    Nanobody, compound or polypeptide of the invention can be determined    in any manner known per se, such as by pharmacokinetic analysis.    Suitable techniques will be clear to the person skilled in the art,    and may for example generally involve the steps of suitably    administering to a warm-blooded animal (i.e. to a human or to    another suitable mammal, such as a mouse, rabbit, rat, pig, dog or a    primate, for example monkeys from the genus Macaca (such as, and in    particular, cynomolgus monkeys (Macaca fascicularis) and/or rhesus    monkeys (Macaca mulatta)) and baboon (Papio ursinus)) a suitable    dose of the amino acid sequence, Nanobody, compound or polypeptide    of the invention; collecting blood samples or other samples from    said animal; determining the level or concentration of the amino    acid sequence, Nanobody, compound or polypeptide of the invention in    said blood sample; and calculating, from (a plot of) the data thus    obtained, the time until the level or concentration of the amino    acid sequence, Nanobody, compound or polypeptide of the invention    has been reduced by 50% compared to the initial level upon dosing.    Reference is for example made to the Experimental Part below, as    well as to the standard handbooks, such as Kenneth A et al.    (Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists)    and Peters et al. (1996, Pharmacokinete analysis: A Practical    Approach). Reference is also made to Gibaidi M and Perron D (1982,    “Pharmacokinetics”, published by Marcel Dekker, 2nd Rev. edition).    -   As will also be clear to the skilled person (see for example        pages 6 and 7 of WO 04/003019 and in the further references        cited therein), the half-life can be expressed using parameters        such as the t½-alpha, t½-beta and the area under the curve        (AUC). In the present specification, an “increase in half-life”        refers to an increase in any one of these parameters, such as        any two of these parameters, or essentially all three these        parameters. As used herein “increase in half-life” or “increased        half-life” in particular refers to an increase in the t½-beta,        either with or without an increase in the t½-alpha and/or the        AUC or both.-   p) In the context of the present invention, “modulating” or “to    modulate” generally means either reducing or inhibiting the activity    of, or alternatively increasing the activity of, a target or    antigen, as measured using a suitable in vitro, cellular or in vivo    assay. In particular, “modulating” or “to modulate” may mean either    reducing or inhibiting the activity of, or alternatively increasing    a (relevant or intended) biological activity of, a target or    antigen, as measured using a suitable in vitro, cellular or in vivo    assay (which will usually depend on the target or antigen involved),    by at least 1%, preferably at least 5%, such as at least 10% or at    least 25%, for example by at least 50%, at least 60%, at least 70%,    at least 80%, or 90% or more, compared to activity of the target or    antigen in the same assay under the same conditions but without the    presence of the amino acid sequence, Nanobody, polypeptide, compound    or construct of the invention.    -   As will be clear to the skilled person, “modulating” may also        involve effecting a change (which may either be an increase or a        decrease) in affinity, avidity, specificity and/or selectivity        of a target or antigen for one or more of its ligands, binding        partners, partners for association into a homomultimeric or        heteromultimeric form, or substrates; and/or effecting a change        (which may either be an increase or a decrease) in the        sensitivity of the target or antigen for one or more conditions        in the medium or surroundings in which the target or antigen is        present (such as pH, ion strength, the presence of co-factors,        etc.), compared to the same conditions but without the presence        of the amino acid sequence, Nanobody, polypeptide, compound or        construct of the invention. As will be clear to the skilled        person, this may again be determined in any suitable manner        and/or using any suitable assay known per se, depending on the        target or antigen involved.    -   “Modulating” may also mean effecting a change (i.e. an activity        as an agonist, as an antagonist or as a reverse agonist,        respectively, depending on the target or antigen and the desired        biological or physiological effect) with respect to one or more        biological or physiological mechanisms, effects, responses,        functions, pathways or activities in which the target or antigen        (or in which its substrate(s), ligand(s) or pathway(s) are        involved, such as its signalling pathway or metabolic pathway        and their associated biological or physiological effects) is        involved. Again, as will be clear to the skilled person, such an        action as an agonist or an antagonist may be determined in any        suitable manner and/or using any suitable (in vitro and usually        cellular or in assay) assay known per se, depending on the        target or antigen involved. In particular, an action as an        agonist or antagonist may be such that an intended biological or        physiological activity is increased or decreased, respectively,        by at least 1%, preferably at least 5%, such as at least 10% or        at least 25%, for example by at least 50%, at least 60%, at        least 70%, at least 80%, or 90% or more, compared to the        biological or physiological activity in the same assay under the        same conditions but without the presence of the amino acid        sequence, Nanobody, polypeptide, compound or construct of the        invention.    -   Modulating may for example also involve allosteric modulation of        the target or antigen; and/or reducing or inhibiting the binding        of the target or antigen to one of its substrates or ligands        and/or competing with a natural ligand, substrate for binding to        the target or antigen. Modulating may also involve activating        the target or antigen or the mechanism or pathway in which it is        involved. Modulating may for example also involve effecting a        change in respect of the folding or confirmation of the target        or antigen, or in respect of the ability of the target or        antigen to fold, to change its confirmation (for example, upon        binding of a ligand), to associate with other (sub)units, or to        disassociate. Modulating may for example also involve effecting        a change in the ability of the target or antigen to transport        other compounds or to serve as a channel for other compounds        (such as ions).    -   Modulating may be reversible or irreversible, but for        pharmaceutical and pharmacological purposes will usually be in a        reversible manner.    -   In the context of the present invention, “modulating” or “to        modulate” generally means exercising an agonistic or        antagonistic effect, respectively, with respect to IL-6, IL-6R        and/or the biological pathways, responses, signalling,        mechanisms or effects in which IL-6 and/or IL-6R is involved. In        particular, “modulating” or “to modulate” may mean either an        agonistic or antagonistic effect (i.e. a full or partial        agonistic or antagonistic effect, respectively), as measured        using a suitable in vitro, cellular or in vivo assay (such as        those mentioned herein), that leads to a change in a relevant        parameter by at least 1%, preferably at least 5%, such as at        least 10% or at least 25%, for example by at least 50%, at least        60%, at least 70%, at least 80%, or 90% or more, compared to the        same parameter in the same assay under the same conditions but        without the presence of the amino acid sequence, Nanobody,        polypeptide, compound or construct of the invention.    -   In the context of the present invention “modulating, inhibiting        and/or preventing binding of the IL-6/IL-6R complex to gp130”        means that the amino acid sequences, Nanobodies, polypeptides,        compounds or constructs of the present invention bind to the        specific epitope on IL-6R (i.e. as such or as present in the        IL-6/IL-6R complex) in such a way that the formation of the        IL-6/IL-6R complex is affected, inhibited and/or prevented (e.g.        fully or partially disrupted) in such a way that the binding of        the complex to—e.g. its affinity for—gp130 is reduced, inhibited        and/or prevented (or reversely, that the binding of gp 130        to—e.g. its affinity for—the complex is reduced, inhibited        and/or prevented), so that the signaling induced/mediated by the        binding of the complex to gp130 is modulated (e.g. reduced,        inhibited and/or prevented) compared to the formation of the        complex and its binding to gp130 without the presence of the        amino acid sequence, Nanobodies, polypeptide, compound or        construct of the invention.-   q) in respect of a target or antigen, the term “interaction site” on    the target or antigen means a site, epitope, antigenic determinant,    part, domain or stretch of amino acid residues on the target or    antigen that is a site for binding to a ligand, receptor or other    binding partner, a catalytic site, a cleavage site, a site for    allosteric interaction, a site involved in multimerisation (such as    homomerization or heterodimerization) of the target or antigen; or    any other site, epitope, antigenic determinant, part, domain or    stretch of amino acid residues on the target or antigen that is    involved in a biological action or mechanism of the target or    antigen. More generally, an “interaction site” can be any site,    epitope, antigenic determinant, part, domain or stretch of amino    acid residues on the target or antigen to which an amino acid    sequence, Nanobody, polypeptide, compound or construct of the    invention can bind such that the target or antigen (and/or any    pathway, interaction, signalling, biological mechanism or biological    effect in which the target or antigen is involved) is modulated (as    defined herein).-   r) A “stretch of amino acid residues” means two or more amino acid    residues that are adjacent to each other or in close proximity to    each other, i.e. in the primary or tertiary structure of the amino    acid sequence. In the context of the present invention, the “stretch    of amino acid residues” will be (at least partially) responsible for    the binding of the amino acid sequence, Nanobody, polypeptide,    compound or construct of the invention to its specific epitope on    IL-6R.-   s) An amino acid sequence, Nanobody, polypeptide, compound or    construct is said to be “specific for” a first target or antigen    compared to a second target or antigen when it binds to the first    antigen with an affinity (as described above, and suitably expressed    as a K_(D) value, K_(A) value, K_(off) rate and/or K_(on) rate) that    is at least 10 times, such as at least 100 times, and preferably at    least 1000 times, and up to 10.000 times or more better than the    affinity with which said amino acid sequence, Nanobody, polypeptide,    compound or construct binds to the second target or polypeptide. For    example, the first amino acid sequence, Nanobody, polypeptide,    compound or construct may bind to the target or antigen with a K_(D)    value that is at least 10 times less, such as at least 100 times    less, and preferably at least 1000 times less, such as 10.000 times    less or even less than that, than the K_(D) with which said amino    acid sequence, Nanobody, polypeptide, compound or construct binds to    the second target or polypeptide. Preferably, when an amino acid    sequence, Nanobody, polypeptide, compound or construct is “specific    for” a first target or antigen compared to a second target or    antigen, it is directed against (as defined herein) said first    target or antigen, but not directed against said second target or    antigen.-   t) An amino acid sequence or Nanobody of the invention (as well as    compounds, constructs and polypeptides comprising the same) is    “cross-reactive” with IL-6R from two different species (i.e. a first    species and a second species) means that the amino acid sequence or    Nanobody of the invention (as well as compounds, constructs and    polypeptides comprising the same) binds to IL-6R from a second    species with an affinity (suitably measured and/or expressed as a    K_(D)-value (actual or apparent), a K_(A)-value (actual or    apparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively as    an IC₅₀ value, as further described herein) that is the same or at    least 70% (preferably at least 80%, more preferably at least 90%, or    even more preferably at least 95%) of the affinity with which said    amino acid sequence or Nanobody of the invention (as well as    compounds, constructs and polypeptides comprising the same) binds to    IL-6R from a first species.-   u) As further described herein, the total number of amino acid    residues in a Nanobody can be in the region of 110-120, is    preferably 112-115, and is most preferably 113. It should however be    noted that parts, fragments, analogs or derivatives (as further    described herein) of a Nanobody are not particularly limited as to    their length and/or size, as long as such parts, fragments, analogs    or derivatives meet the further requirements outlined herein and are    also preferably suitable for the purposes described herein;-   v) The amino acid residues of a Nanobody are numbered according to    the general numbering for V_(H) domains given by Kabat et al.    (“Sequence of proteins of immunological interest”, US Public Health    Services, NIH Bethesda, Md., Publication No. 91), as applied to    V_(HH) domains from Camelids in the article of Riechmann and    Muyldermans (2000, J. Immunol. Methods 240 (1-2): 185-195; see for    example FIG. 2 of this publication); or referred to herein.    According to this numbering, FR1 of a Nanobody comprises the amino    acid residues at positions 1-30, CDR1 of a Nanobody comprises the    amino acid residues at positions 31-35, FR2 of a Nanobody comprises    the amino acids at positions 36-49, CDR2 of a Nanobody comprises the    amino acid residues at positions 50-65, FR3 of a Nanobody comprises    the amino acid residues at positions 66-94, CDR3 of a Nanobody    comprises the amino acid residues at positions 95-182, and FR4 of a    Nanobody comprises the amino acid residues at positions 103-113. [In    this respect, it should be noted that—as is well known in the art    for V_(H) domains and for V_(HH) domains—the total number of amino    acid residues in each of the CDR's may vary and may not correspond    to the total number of amino acid residues indicated by the Kabat    numbering (that is, one or more positions according to the Kabat    numbering may not be occupied in the actual sequence, or the actual    sequence may contain more amino acid residues than the number    allowed for by the Kabat numbering). This means that, generally, the    numbering according to Kabat may or may not correspond to the actual    numbering of the amino acid residues in the actual sequence.    Generally, however, it can be said that, according to the numbering    of Kabat and irrespective of the number of amino acid residues in    the CDR's, position 1 according to the Kabat numbering corresponds    to the start of FR1 and vice versa, position 36 according to the    Kabat numbering corresponds to the start of FR2 and vice versa,    position 66 according to the Kabat numbering corresponds to the    start of FR3 and vice versa, and position 103 according to the Kabat    numbering corresponds to the start of FM and vice versa.].    -   Alternative methods for numbering the amino acid residues of        V_(H) domains, which methods can also be applied in an analogous        manner to V_(HH) domains from Camelids and to Nanobodies, are        the method described by Chothia at al. (1989, Nature 342:        877-883), the so-called “AbM definition” and the so-called        “contact definition”, However, in the present description,        claims and figures, the numbering according to Kabat as applied        to V_(HH) domains by Riechmann and Muyldermans will be followed,        unless indicated otherwise; and-   w) The Figures, Sequence Listing and the Experimental Part/Examples    are only given to further illustrate the invention and should not be    interpreted or construed as limiting the scope of the invention    and/or of the appended claims in any way, unless explicitly    indicated otherwise herein.

The present invention provides stretches of amino acid residues (SEQ IDNO's: 80-82, SEQ ID NO's: 84-91 and SEQ ID NO's: 93-95) that areparticularly suited for binding to IL-6R. These stretches of amino acidresidues may be present in, and/or may be incorporated into, an aminoacid sequence of the invention, in particular in such a way that theyform (part of) the antigen binding site of the amino acid sequence ofthe invention. These stretches of amino acid residues have beengenerated as CDR sequences of heavy chain antibodies or V_(HH) sequencesthat were raised against the IL-6R and that were further affinitymatured (see Example section) to further increase their affinity forbinding to IL-6R as well as other properties such as their efficacyand/or potency, and/or their selectivity, in addition to their capacityto partially or totally block the IL-6/IL-6R interaction, and/or inhibitsignalization through, IL-6, IL-6R and/or the IL-6/IL-6R complex. Thesestretches of amino acid residues are also referred to herein as “CDRsequences of the invention” (i.e. as “CDR1 sequences of the invention”,“CDR2 sequences of the invention” and “CDR3 sequences of the invention”,respectively).

It should however be noted that the invention in its broadest sense isnot limited to a specific structural role or function that thesestretches of amino acid residues may have in an amino acid sequence ofthe invention, as long as these stretches of amino acid residues allowthe amino acid sequence of the invention to bind to IL-6R. Thus,generally, the invention in its broadest sense provides amino acidsequences that are capable of binding to IL-6R with a certain specifiedaffinity, avidity, efficacy and/or potency in addition to their capacityto partially or totally block the IL-6/IL-6R interaction, and/or inhibitsignalization through, IL-6, IL-6R and/or the IL-6/IL-6R complex andthat comprises one or more CDR sequences as described herein and, inparticular a suitable combination of two or more such CDR sequences,that are suitably linked to each other via one or more further aminoacid sequences, such that the entire amino acid sequence forms a bindingdomain and/or binding unit that is capable of binding to IL-6R. Itshould however also be noted that the presence of only one such CDRsequence in an amino acid sequence of the invention may by itselfalready be sufficient to provide the amino acid sequence of theinvention the capacity of binding to IL-6R; reference is for exampleagain made to the so-called “Expedite fragments” described in WO03/050531.

Thus, in a specific, but non-limiting aspect, the amino acid sequence ofthe invention may comprises at least one stretch of amino acid residuesthat is chosen from the group consisting of:

-   -   the CDR1 sequences:    -   a) SEQ ID NO's: 80-82; or    -   b) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with one of SEQ        ID NO's: 80-82, provided that the amino acid sequence comprising        said stretch of amino acid residues binds IL-6R with the same,        about the same, or a higher affinity compared to the amino acid        sequence comprising said stretch of amino acid residues without        the 2 or 1 amino acid difference, said affinity as measured by        surface plasmon resonance;    -   and/or    -   the CDR2 sequences:    -   c) SEQ ID NO's: 84-91; or    -   d) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with one of SEQ        ID NO's: 84-91, provided that the amino acid sequence comprising        said stretch of amino acid residues binds IL-6R with the same,        about the same, or a higher affinity compared to the amino acid        sequence comprising said stretch of amino acid residues without        the 2 or 1 amino acid difference, said affinity as measured by        surface plasmon resonance;    -   and/or    -   the CDR3 sequences:    -   e) SEQ ID NO's: 93-95; or    -   f) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with one of SEQ        ID ND's: 93-95, provided that the amino acid sequence comprising        said stretch of amino acid residues binds IL-6R with the same,        about the same or a higher affinity compared to the amino acid        sequence comprising said stretch of amino acid residues without        the 2 or 1 amino acid difference, said affinity as measured by        surface plasmon resonance.

In particular, an amino acid sequence of the invention may be an aminoacid sequence that comprises at least one antigen binding site, whereinsaid antigen binding site comprises at least one stretch of amino acidresidues that is chosen from the group consisting of the CDR1 sequences,CDR2 sequences and CDR3 sequences as described above (or any suitablecombination thereof). In a preferred aspect, however, the amino acidsequence of the invention comprises more than one, such as two or morestretches of amino acid residues chosen from the group consisting of theCDR1 sequences of the invention, the CDR2 sequences of the inventionand/or the CDR3 sequences of the invention.

Accordingly the present invention also relates to amino acid sequencesthat comprises two or more stretches of amino acid residues chosen fromthe following:

-   -   the CDR1 sequences    -   a) SEQ ID NO's: 80-82; or    -   b) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with one of SEQ        ID NO's: 80-82, provided that the amino acid sequence comprising        said stretch of amino acid residues binds IL-6R with the same,        about the same or a higher affinity compared to the amino acid        sequence comprising said stretch of amino acid residues without        the 2 or 1 amino acid difference, said affinity as measured by        surface plasmon resonance;    -   and/or    -   the CDR2 sequences    -   c) SEQ ID NO's: 84-91; or    -   d) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with one of SEQ        ID NO's: 84-91, provided that the amino acid sequence comprising        said stretch of amino acid residues binds IL-6R with the same,        about the same, or a higher affinity compared to the amino acid        sequence comprising said stretch of amino acid residues without        the 2 or 1 amino acid difference, said affinity as measured by        surface plasmon resonance;    -   and/or    -   the CDR3 sequences    -   e) SEQ ID NO's: 93-95; or    -   f) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with one of SEQ        ID NO's: 93-95, provided that the amino acid sequence comprising        said stretch of amino acid residues binds IL-6R with the same,        about the same, or a higher affinity compared to the amino acid        sequence comprising said stretch of amino acid residues without        the 2 or 1 amino acid difference, said affinity as measured by        surface plasmon resonance.    -   such that (i) when the first stretch of amino acid residues        corresponds to one of the amino acid sequences according to a),        or b), the second stretch of amino acid residues corresponds to        one of the amino acid sequences according to c), d), e) or        f); (ii) when the first stretch of amino acid residues        corresponds to one of the amino acid sequences according to c)        or d), the second stretch of amino acid residues corresponds to        one of the amino acid sequences according to a), b), e) or f);        or (iii) when the first stretch of amino acid residues        corresponds to one of the amino acid sequences according to e)        or f), the second stretch of amino acid residues corresponds to        one of the amino acid sequences according to a), b), c) or d).

In a specific aspect, the present invention also relates to amino acidsequences that comprises three or more stretches of amino acid residues,in which the first stretch of amino acid residues is chosen from thefollowing CDR1 sequences:

-   -   a) SEQ ID NO's: 80-82; or    -   b) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with one of SEQ        ID NO's: 80-82, provided that the amino acid sequence comprising        said stretch of amino acid residues binds IL-6R with the same,        about the same, or a higher affinity compared to the amino acid        sequence comprising said stretch of amino acid residues without        the 2 or 1 amino acid difference, said affinity as measured by        surface plasmon resonance;    -   the second stretch of amino acid residues is chosen from the        following CDR2 sequences:    -   c) SEQ ID NO's: 84-91; or    -   d) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with one of SEQ        ID NO's: 84-91, provided that the amino acid sequence comprising        said stretch of amino acid residues binds IL-6R with the same,        about the same, or a higher affinity compared to the amino acid        sequence comprising said stretch of amino acid residues without        the 2 or 1 amino acid difference, said affinity as measured by        surface plasmon resonance;    -   and the third stretch of amino acid residues is chosen from the        following CDR3 sequences:    -   e) SEQ ID NO's: 93-95; or    -   f) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with one of SEQ        ID NO's: 93-95, provided that the amino acid sequence comprising        said stretch of amino acid residues binds IL-6R with the same,        about the same, or a higher affinity compared to the amino acid        sequence comprising said stretch of amino acid residues without        the 2 or 1 amino acid difference, said affinity as measured by        surface plasmon resonance.

As described herein, the present invention also encompasses amino acidsequences that comprise one or more stretches of amino acid residuesthat have no more than 2, preferably no more than 1 amino aciddifference with one of the stretches of amino acid residues specified ina), c) and/or e), i.e. with one of the specified CDR1 sequences (i.e.with one of SEQ ID NO's: 80-82), with one of the specified CDR2sequences (i.e. with one of SEQ ID NO's: 84-91) and/or with one of thespecified CDR3 sequences (i.e. with one of SEQ ID NO's: 93-95).

The term “amino acid difference” refers to an insertion, deletion orsubstitution of a single amino acid residue on a position of the stretchof amino acid residues (or CDR sequence) specified in b), d) or f),compared to the stretch of amino acid residues (or CDR sequence) ofrespectively a), c) or e); it being understood that the stretch of aminoacid residues (or CDR sequence) of b), d) and f) can contain one ormaximal two such amino acid differences compared to the stretch of aminoacid residues of respectively a), c) or e).

The “amino acid difference” can be any one or maximal two substitutions,deletions or insertions, or any combination thereof, that either improvethe properties of the amino acid sequence of the invention or that atleast do not detract too much from the desired properties or from thebalance or combination of desired properties of the amino acid sequenceof the invention. In this respect, the resulting amino acid sequence ofthe invention should at least bind IL-6R with the same, about the same,or a higher affinity compared to the amino acid sequence comprising theone or more stretches of amino acid residues without the one or maximaltwo substitutions, deletions or insertions, said affinity as measured bysurface plasmon resonance.

For example, and depending on the host organism used to express theamino acid sequence of the invention, such deletions and/orsubstitutions may be designed in such a way that one or more sites forpost-translational modification (such as one or more glycosylationsites) are removed, as will be within the ability of the person skilledin the art.

In a preferred aspect of the invention, the “amino acid difference” isan amino acid substitution. The amino acid substitution may be any oneor maximal two substitutions that either improve the properties of theamino acid sequence of the invention or that at least do not detract toomuch from the desired properties or from the balance or combination ofdesired properties of the amino acid sequence of the invention. In thisrespect, the resulting amino acid sequence of the invention should atleast bind IL-6R with the same, about the same, or a higher affinitycompared to the amino acid sequence comprising the one or more stretchesof amino acid residues without the one or maximal two substitutions,said affinity as measured by surface plasmon resonance.

The amino acid substitution in the one or more stretches of amino acidresidues may be a conservative amino acid substitution. “Conservative”amino acid substitutions are generally amino acid substitutions in whichan amino acid residue is replaced with another amino acid residue ofsimilar chemical structure and which has little or essentially noinfluence on the function, activity or other biological properties ofthe resulting amino acid sequence. Such conservative amino acidsubstitutions are well known in the art, for example from WO 04/037999,GB 3357768-A, WO 98/49185, WO 00/46383 and WO 01/09300; and (preferred)types and/or combinations of such substitutions may be selected on thebasis of the pertinent teachings from WO 04/037999 as well as WO98/49185 and from the further references cited therein.

Such conservative substitutions preferably are substitutions in whichone amino acid within the following groups (a)-(e) is substituted byanother amino acid residue within the same group: (a) small aliphatic,nonpolar or slightly polar residues: Ala, Ser, Thr, Pro and Gly; (b)polar, negatively charged residues and their (uncharged) amides: Asp,Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg andLys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val and Cys;and (e) aromatic residues: Phe, Tyr and Trp.

Particularly preferred conservative substitutions are as follows: Alainto Gly or into Ser; Arg into Lys; Asn into Gln or into His; Asp intoGlu; Cys into Ser; Gln into Asn; Glu into Asp; Gly into Ala or into Pro;His into Asn or into Gln; Ile into Leu or into Val; Leu into Ile or intoVal; Lys into Arg, into Gln or into Glu; Met into Leu, into Tyr or intoIle; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trpinto Tyr; Tyr into Trp; and/or Phe into Val, into Ile or into Leu.

In another aspect of the invention, the amino acid substitutions in theone or more stretches of amino acid residues may provides the amino acidsequence with increased affinity for binding to IL-6R. This may be doneby techniques such as random or site-directed mutagenesis and/or othertechniques for affinity maturation known per se, such as e.g. describedin WO 09/004,065, WO 2009/004066, WO 05/003345, WO 06/023144, EP 527809,EP 397834.

Without being limiting, rules (partly or fully followed) forsubstitutions of amino acid residues in the CDRs may be as follows (i.e.substitution with amino acids with similar side chain chemistries):

K is substituted by R;

R is substituted by K;

A is substituted by S or T;

S is substituted by A or T;

T is substituted by A or S;

I is substituted by L or V;

L is substituted by I or V;

V is substituted by I or L;

F is substituted by Y;

Y is substituted by F;

N is substituted by D;

D is substituted by N;

is substituted by E;

E is substituted by Q;

G is substituted by A;

M is substituted by L;

H, C, W and P are kept constant.

Furthermore, and also without being limiting, the rules (partly or fullyfollowed) for substitutions of amino acid residues in the CDRs may bealternatively as follows for substitutions at positions 27 to 35 andpositions 50 to 5S (using Kabat numbering system), wherein for positions27 to 35:

-   -   Original amino acid residue in position 27 (Kabat numbering        used) is substituted by F; G; R; S; 2 out of F, G, R, S; 3 out        of F, G, R, S; or all of them, preferably all of them;    -   Original amino acid residue in position 28 (Kabat numbering        used) is substituted by A; I; S; T; 2 out of A, I, S, T; 3 out        of A, I, S, T; or all of them, preferably all of them;    -   Original amino acid residue in position 29 (Kabat numbering        used) is substituted by F; G; L; S; 2 out of F, G, L, S; 3 out        of F, G, L, S; or all of them, preferably all of them;    -   Original amino acid residue in position 30 (Kabat numbering        used) is substituted by D; G; S; T; 2 out of D, G, S, T; 3 out        of D, G, S, T; or all of them, preferably all of them;    -   Original amino acid residue in position 31 (Kabat numbering        used) is substituted by D; I; N; S; T; 2 out of D, I, N, S, T; 3        out of D, I, N, S, T; or all of them, preferably all of them;    -   Original amino acid residue in position 32 (Kabat numbering        used) is substituted by D; N; Y; 2 out of D, n, Y; or all of        them, preferably all of them;    -   Original amino acid residue in position 33 (Kabat numbering        used) is substituted by A; G; T; V; 2 out of A, G, T, V; 3 out        of A, G, T, V; or all of them, preferably all of them;    -   Original amino acid residue in position 34 (Kabat numbering        used) is substituted by I; M; or all of them, preferably all of        them;    -   Original amino acid residue in position 35 (Kabat numbering        used) is substituted by A; G; S; 2 out of A, G, S; or all of        them, preferably all of them;        and positions 50 to 58 if original amino acid sequence has an        amino acid sequence in position 52a (Kabat numbering used),    -   Original amino acid residue in position 50 (Kabat numbering        used) is substituted by A; C; G; S; T; 2 out of A, C, G, S, T; 3        out of A, C, G, S, T; 4 out of A, C, G, S, T; or all of them,        preferably all of them;    -   Original amino acid residue in position 51 (Kabat numbering        used) is substituted by I;    -   Original amino acid residue in position 52 (Kabat numbering        used) is substituted by N; R; S; T; 2 out of N, R, S, T; 3 out        of N, R, S, T; or all of them, preferably all of them;    -   Original amino acid residue in position 52a (Kabat numbering        used) is substituted by R; S; T; W; 2 out of R, S, T, W; 3 out        of R, S, T, W; or all of them, preferably all of them;    -   Original amino acid residue in position 53 (Kabat numbering        used) is substituted by D; G; N; S; T; 2 out of D, G, N, S, T; 3        out of D, G, N, S, T; 4 out of D, G, N, S, T; or all of them,        preferably all of them;    -   Original amino acid residue in position 54 (Kabat numbering        used) is substituted by D; G; or all of them, preferably all of        them;    -   Original amino acid residue in position 55 (Kabat numbering        used) is substituted by D; G; S; 2 out of D, G, S; or all of        them, preferably all of them;    -   Original amino acid residue in position 56 (Kabat numbering        used) is substituted by I; N; R; S; T; 2 out of I, N, R, S, T; 3        out of I, N, R, S, T; 4 out of I, N, R, S, T; or all of them,        preferably all of them;    -   Original amino acid residue in position 57 (Kabat numbering        used) is substituted by T;    -   Original amino acid residue in position 58 (Kabat numbering        used) is substituted by D; H; N; S; Y; 2 out of D, H, N, S, Y; 3        out of D, H, N, S, Y; 4 out of D, H, N, S, Y; or all of them,        preferably all of them;        and wherein for positions 50 to 58 if original amino acid        sequence has not an amino acid sequence in position 52a (Kabat        numbering used),    -   Original amino acid residue in position 50 (Kabat numbering        used) is substituted by A; G; R; S; T; 2 out of A, G, R, S, T; 3        out of A, G, R, S, T; 4 out of A, G, R, S, T; or all of them,        preferably all of them;    -   Original amino acid residue in position 51 (Kabat numbering        used) is substituted by I;    -   Original amino acid residue in position 52 (Kabat numbering        used) is substituted by N; S; T; 2 out of N, S, T; or all of        them, preferably all of them;    -   Original amino acid residue in position 53 (Kabat numbering        used) is substituted by N; R; S; T; Y; 2 out of N, R, S, T, Y; 3        out of N, R, S, T, Y; 4 out of N, R, S, T, Y; or all of them,        preferably all of them;    -   Original amino acid residue in position 54 (Kabat numbering        used) is substituted by D; G; R; S; 2 out of D, G, R, S; 3 out        of D, G, R, S; or all of them, preferably all of them;    -   Original amino acid residue in position 55 (Kabat numbering        used) is substituted by G;    -   Original amino acid residue in position 56 (Kabat numbering        used) is substituted by G; N; R; S; T; 2 out of O, N, R, S, T; 3        out of D, N, R, S, T; 4 out of O, N, R, S, T; or all of them,        preferably all of them;    -   Original amino acid residue in position 57 (Kabat numbering        used) is substituted by T;    -   Original amino acid residue in position 58 (Kabat numbering        used) is substituted by D; N; T; Y; 2 out of D, N, T, Y; 3 out        of O, N, T, Y; or all of them, preferably all of them.        after which one or more of the potentially useful substitutions        (or combinations thereof) thus determined can be introduced into        said CDR sequence (in any manner known per se, as further        described herein) and the resulting amino acid sequence(s) can        be tested for affinity for IL-6R, and/or for other desired        properties such as the capacity to (partially or preferably        totally) block the IL-6/IL-6R interaction and/or inhibit        signalization through, IL-6, IL-6R and/or the IL-6/IL-6R        complex. In this way, by means of a limited degree of trial and        error, other suitable substitutions in the CDRs (or suitable        combinations thereof) can be determined by the skilled person        based on the disclosure herein.

The amino acid sequence of the invention may be any amino acid sequencethat comprises at least one stretch of amino acid residues, in whichsaid stretch of amino acid residues has an amino acid sequence thatcorresponds to the sequence of at least one of the CDR sequences definedherein. Such an amino acid sequence may or may not comprise animmunoglobulin fold. For example, and without limitation, such an aminoacid sequence may be a suitable fragment of an immunoglobulin sequencethat comprises at least one such CDR sequence (as defined above), butthat is not large enough to form a (complete) immunoglobulin fold(reference is for example again made to the “Expedite fragments”described in WO 03/050531). Alternatively, such an amino acid sequencemay be a suitable “protein scaffold” that comprises at least one stretchof amino acid residues that corresponds to a CDR sequence as definedherein for the amino acid sequences of the invention (i.e. as part ofits antigen binding site). Suitable scaffolds for presenting amino acidsequences will be clear to the skilled person, and for example comprise,without limitation, to binding scaffolds based on or derived fromimmunoglobulins (i.e. other than the immunoglobulin sequences alreadydescribed herein), protein scaffolds derived from protein A domains(such as Affibodies™), tendamistat, fibronectin, lipocalin, CTLA-4,T-cell receptors, designed ankyrin repeats, avimers and PDZ domains(Binz et al., 2005, Nat. Biotech., 23: 1257), and binding moieties basedon DNA or RNA including but not limited to DNA or RNA aptamers (Ulrichet al., 2006, Comb. Chem. High Throughput Screen 9(8): 619-32).

Again, any amino acid sequence of the invention that comprises one ormore of the CDR sequences as defined herein for the amino acid sequencesof the invention (i.e. “CDR of the invention”) is preferably such thatit can specifically bind (as defined herein) to IL-6R, and more inparticular such that it can bind to IL-6R with an affinity (suitablymeasured and/or expressed as a K_(D)-value (actual or apparent), aK_(A)-value (actual or apparent), a k_(on)-rate and/or a k_(off)-rate,or alternatively as an IC₅₀ value, as further described herein), that isas defined herein. Any amino acid sequence of the invention thatcomprises one or more of the CDR sequences as defined herein for theamino acid sequences of the invention is preferably such that it has acell based potency and a plasma potency as defined herein.

Furthermore, it will also be clear to the skilled person that it may bepossible to “graft” one or more of the CDR's defined herein for theamino acid sequences of the invention (i.e. “CDR of the invention”) ontoother “scaffolds”, including but not limited to human scaffolds ornon-immunoglobulin scaffolds. Suitable scaffolds and techniques for suchCDR grafting will be clear to the skilled person and are well known inthe art, see for example U.S. Pat. No. 7,180,370, WO 01/27160, EP0605522, EP 0460167, U.S. Pat. No. 7,054,297, Nicaise et al. (2004,Protein Science, 13: 1882-1891), Ewert et al. (2004, Methods, 34(2):184-199), Kettleborough et al. (1991, Protein Eng. 4(7): 773-783),O'Brien and Jones (2003, Methods Mol. Biol. 207: 81-100), Skerra (2000,J. Mol. Recognit. 13: 167-187), and Saerens et al. (2005, J. Mol. Biol.352(3): 597-607), and the further references cited therein. For example,techniques known per se for grafting mouse or rat CDR's onto humanframeworks and scaffolds can be used in an analogous manner to providechimeric proteins comprising one or more of the CDR sequences definedherein for the amino acid sequences of the invention and one or morehuman framework regions or sequences.

Thus, in a specific aspect, the invention also encompasses chimericamino acid sequences comprising at least one CDR sequence chosen fromthe group consisting of CDR1 sequences of the invention, CDR2 sequencesof the invention and CDR3 sequences of the invention (defined herein forthe amino acid sequences of the invention). Preferably, such a chimericamino acid sequences comprise at least one CDR sequence chosen from thegroup consisting of the CDR1 sequences of the invention (defined hereinfor the amino acid sequences of the invention), and also at least oneCDR sequence chosen from the group consisting of the CDR2 sequences ofthe invention (defined herein for the amino acid sequences of theinvention); or at least one CDR sequence chosen from the groupconsisting of the CDR1 sequences of the invention (defined herein forthe amino acid sequences of the invention) and at least one CDR sequencechosen from the group consisting of the CDR3 sequences of the invention(defined herein for the amino acid sequences of the invention); or sucha chimeric polypeptide may comprises at least one CDR sequence chosenfrom the group consisting of the CDR2 sequences of the invention(defined herein for the amino acid sequences of the invention) and alsoat least one CDR sequence chosen from the group consisting of the CDR3sequences of the invention (defined herein for the amino acid sequencesof the invention). For example, such a chimeric polypeptide may compriseone CDR sequence chosen from the group consisting of the CDR3 sequencesof the invention (defined herein for the amino acid sequences of theinvention), one CDR sequence chosen from the group consisting of theCDR1 sequences of the invention (defined herein for the amino acidsequences of the invention) and one CDR sequence chosen from the groupconsisting of the CDR2 sequences of the invention (defined herein forthe amino acid sequences of the invention). The combinations of CDR'sthat are mentioned herein as being preferred for the amino acidsequences of the invention (see Table A-1) will usually also bepreferred for these chimeric polypeptides.

In said chimeric polypeptides, the CDR's may be linked to further aminoacid sequences sequences and/or may be linked to each other via aminoacid sequences, in which said amino acid sequences are preferablyframework sequences or are amino acid sequences that act as frameworksequences, or together form a scaffold for presenting the CDR's.

According to one non-limiting embodiment, the chimeric amino acidsequences comprises at least two CDR sequences (defined herein for theamino acid sequences of the invention) linked via at least one frameworksequence, in which preferably at least one of the two CDR sequences is aCDR3 sequence, with the other CDR sequence being a CDR1 or CDR2sequence. According to a preferred, but non-limiting embodiment, thechimeric amino acid sequences comprise at least three CDR sequences ofthe invention (defined herein for the amino acid sequences of theinvention) linked to at least two framework sequences, in whichpreferably at least one of the three CDR sequences is a CDR3 sequence,with the other two CDR sequences being CDR1 or CDR2 sequences, andpreferably being one CDR1 sequence and one CDR2 sequence. According toone specifically preferred, but non-limiting embodiment, the chimericamino acid sequences have the structureFR1′-CDR1-FR2′-CDR2-FR3′-CDR3-FR4′, in which CDR1, CDR2 and CDR3 are asdefined herein for the amino acid sequences of the invention, and FR1′,FR2′, FR3′ and FR4′ are framework sequences. FR1′, FR2′, FR3′ and FR4′may in particular be Framework 1, Framework 2, Framework 3 and Framework4 sequences, respectively, of a human antibody (such as V_(H)3sequences) and/or parts or fragments of such Framework sequences. It isalso possible to use parts or fragments of a chimeric polypeptide withthe structure FR1′-CDR1-FR2′-CDR2-FR3′-CDR3-FR4′. Preferably, such partsor fragments are such that they meet the criteria set out for the aminoacid sequences of the invention.

In such an amino acid sequence of the invention, the framework sequencesmay be any suitable framework sequences, and examples of suitableframework sequences will be clear to the skilled person, for example onthe basis the standard handbooks and the further disclosure and priorart mentioned herein.

The framework sequences are preferably (a suitable combination of)immunoglobulin framework sequences or framework sequences that have beenderived from immunoglobulin framework sequences (for example, bysequence optimization such as humanization or camelization). Forexample, the framework sequences may be framework sequences derived froma light chain variable domain (e.g. a V_(L)-sequence) and/or from aheavy chain variable domain (e.g. a V_(H)-sequence). In one particularlypreferred aspect, the framework sequences are either framework sequencesthat have been derived from a V_(HH)-sequence (in which said frameworksequences may optionally have been partially or fully humanized) or areconventional V_(H) sequences that have been camelized (as definedherein).

The framework sequences may preferably be such that the amino acidsequence of the invention is a domain antibody (or an amino acidsequence that is suitable for use as a domain antibody); is a singledomain antibody (or an amino acid sequence that is suitable for use as asingle domain antibody); is a “dAb” (or an amino acid sequence that issuitable for use as a dAb); or is a Nanobody (including but not limitedto V_(HH) sequence). Again, suitable framework sequences will be clearto the skilled person, for example on the basis the standard handbooksand the further disclosure and prior art mentioned herein.

In particular, the framework sequences present in the amino acidsequences of the invention may contain one or more of Hallmark residues(as defined in WO 08/020,079 (Tables A-3 to A-8)), such that the aminoacid sequence of the invention is a Nanobody. Some preferred, butnon-limiting examples of (suitable combinations of) such frameworksequences will become clear from the further disclosure herein (see e.g.Table A-1). Generally, Nanobodies (in particular V_(HH) sequences and(partially) humanized VHH sequences) can in particular be characterizedby the presence of one or more “Hallmark residues” in one or more of theframework sequences (as e.g. further described in WO 08/020,079, page61, line 24 to page 98, line 3).

In a preferred aspect, the amino acid sequence of the inventioncomprises an immunoglobulin fold or is capable, under suitableconditions to form an immunoglobulin fold. Preferably the amino acidsequence of the invention is an immunoglobulin sequence; and even morepreferably the amino acid sequence of the invention has the structure of

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

Accordingly, the present invention also relates to an amino acidsequence which essentially consists of 4 framework regions (FR1 to FR4,respectively) and 3 complementarity determining regions (CDR1 to CDR3,respectively), in which:

-   -   CDR1 is chosen from the group consisting of:    -   a) SEQ ID NO's: 80-82; or    -   b) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with one of SEQ        ID NO's: 80-82, provided that the amino acid sequence comprising        said stretch of amino acid residues binds IL-6R with the same,        about the same, or a higher affinity compared to the amino acid        sequence comprising said stretch of amino acid residues without        the 2 or 1 amino acid difference, said affinity as measured by        surface plasmon resonance;    -   and/or    -   CDR2 is chosen from the group consisting of:    -   c) SEQ ID NO's: 84-91; or    -   d) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with one of SEQ        ID ND's: 84-91, provided that the amino acid sequence comprising        said stretch of amino acid residues binds IL-6R with the same,        about the same, or a higher affinity compared to the amino acid        sequence comprising said stretch of amino acid residues without        the 2 or 1 amino acid difference, said affinity as measured by        surface plasmon resonance;    -   and/or    -   CDR3 is chosen from the group consisting of:    -   e) SEQ ID NO's: 93-95; or    -   f) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with one of SEQ        ID NO's: 93-95, provided that the amino acid sequence comprising        said stretch of amino acid residues binds IL-6R with the same,        about the same, or a higher affinity compared to the amino acid        sequence comprising said stretch of amino acid residues without        the 2 or 1 amino acid difference, said affinity as measured by        surface plasmon resonance.

In this embodiment, the amino acid sequences comprise at least one CDRsequence chosen from the group consisting of the CDR1 sequences of theinvention (defined herein for the amino acid sequences of theinvention), the CDR2 sequences of the invention (defined herein for theamino acid sequences of the invention), or the CDR3 sequences of theinvention (defined herein for the amino acid sequences of theinvention). Preferably the amino acid sequences comprise at least twoCDR sequence chosen from the group consisting of the CDR1 sequences ofthe invention (defined herein for the amino acid sequences of theinvention), the CDR2 sequences of the invention (defined herein for theamino acid sequences of the invention), or the CDR3 sequences of theinvention (defined herein for the amino acid sequences of theinvention), such as at least one CDR sequence chosen from the groupconsisting of the CDR1 sequences of the invention (defined herein forthe amino acid sequences of the invention) and at least one CDR sequencechosen from the group consisting of the CDR2 sequences of the invention(defined herein for the amino acid sequences of the invention); or atleast one CDR sequence chosen from the group consisting of the CDR1sequences of the invention (defined herein for the amino acid sequencesof the invention) and at least one CDR sequence chosen from the groupconsisting of the CDR3 sequences of the invention (defined herein forthe amino acid sequences of the invention); or at least one CDR sequencechosen from the group consisting of the CDR2 sequences of the invention(defined herein for the amino acid sequences of the invention) and atleast one CDR sequence chosen from the group consisting of the CDR3sequences of the invention (defined herein for the amino acid sequencesof the invention); or such a amino acid sequence may comprises three CDRsequence chosen from the group consisting of the CDR1 sequences of theinvention (defined herein for the amino acid sequences of theinvention), CDR2 sequences of the invention (defined herein for theamino acid sequences of the invention) and the CDR3 sequences of theinvention (defined herein for the amino acid sequences of theinvention). The invention thus also relates to an amino acid sequencewhich essentially consists of 4 framework regions (FR1 to FR4,respectively) and 3 complementarity determining regions (CDR1 to CDR3,respectively), in which:

-   -   CDR1 is chosen from the group consisting of:    -   a) SEQ ID NO's: 80-82; or    -   b) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with one of SEQ        ID NO's: 80-82, provided that the amino acid sequence comprising        said stretch of amino acid residues binds IL-6R with the same,        about the same, or a higher affinity compared to the amino acid        sequence comprising said stretch of amino acid residues without        the 2 or 1 amino acid difference, said affinity as measured by        surface plasmon resonance;    -   and    -   CDR2 is chosen from the group consisting of:    -   c) SEQ ID ND's: 84-91; or    -   d) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with one of SEQ        ID NO's: 84-91, provided that the amino acid sequence comprising        said stretch of amino acid residues binds IL-6R with the same,        about the same, or a higher affinity compared to the amino acid        sequence comprising said stretch of amino acid residues without        the 2 or 1 amino acid difference, said affinity as measured by        surface plasmon resonance;    -   and    -   CDR3 is chosen from the group consisting of:    -   e) SEQ ID NO's: 93-95; or    -   f) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with one of SEQ        ID NO's: 93-95, provided that the amino acid sequence comprising        said stretch of amino acid residues binds IL-6R with the same,        about the same, or a higher affinity compared to the amino acid        sequence comprising said stretch of amino acid residues without        the 2 or 1 amino acid difference, said affinity as measured by        surface plasmon resonance.

Preferred combinations of CDR sequences for the amino acid sequences ofthe invention are shown in Table A-1.

The amino acid sequences of the invention may essentially consists of aheavy chain variable domain sequence that is derived from a conventionalfour-chain antibody or may essentially consist of a heavy chain variabledomain sequence that is derived from heavy chain antibody. The aminoacid sequences of the invention may essentially consists of a domainantibody (or an amino acid sequence that is suitable for use as a domainantibody), of a single domain antibody (or an amino acid sequence thatis suitable for use as a single domain antibody), of a “dAb” (or anamino acid sequence that is suitable for use as a dAb) or of a Nanobody.

For a general description of (single) domain antibodies, reference isalso made to the prior art cited above, as well as to EP 0368684. Forthe term “dAb's”, reference is for example made to Ward et al. (1989,Nature 341: 544-6), to Holt et al. (2003, Trends Biotechnol. 21:484-490); as well as to for example WO 06/030220, WO 06/003388 and otherpublished patent applications of Domantis Ltd. It should also be notedthat, although less preferred in the context of the present inventionbecause they are not of mammalian origin, single domain antibodies orsingle variable domains can be derived from certain species of shark(for example, the so-called “IgNAR domains”, see for example WO05/18629).

In particular, the amino acid sequence of the invention essentiallyconsists of or may be a Nanobody® (as defined herein) or a suitablefragment thereof. [Note: Nanobody®, Nanobodies® and Nanoclone® areregistered trademarks of Ablynx N.V.] Such Nanobodies directed againstIL-6R will also be referred to herein as “Nanobodies of the invention”.

For a general description of Nanobodies, reference is made to thefurther description below, as well as to the prior art cited herein,such as e.g. described in WO 08/020,079 (page 16).

In a specific aspect, the amino acid sequence or Nanobody of theinvention comprises at least SEQ ID NO: 80; or a stretch of amino acidresidues that has no more than 2, preferably no more than 1 amino aciddifference with SEQ ID NO: 80, provided that the amino acid sequencecomprising said stretch of amino acid residues binds IL-6R with thesame, about the same, or a higher affinity compared to the amino acidsequence comprising said stretch of amino acid residues without the 2 or1 amino acid difference, said affinity as measured by surface plasmonresonance.

In another specific aspect, the amino acid sequence or Nanobody of theinvention comprises at least a stretch of amino acid residues chosenfrom SEQ ID NO's: 84, 89 or 91; or a stretch of amino acid residues thathas no more than 2, preferably no more than 1 amino acid difference withone of SEQ ID NO's: 84, 89 or 91, provided that the amino acid sequencecomprising said stretch of amino acid residues binds IL-6R with thesame, about the same, or a higher affinity compared to the amino acidsequence comprising said stretch of amino acid residues without the 2 or1 amino acid difference, said affinity as measured by surface plasmonresonance.

In yet another specific aspect, the amino acid sequence or Nanobody ofthe invention comprises at least SEQ ID NO: 84; or a stretch of aminoacid residues that has no more than 2, preferably no more than 1 aminoacid difference with SEQ ID NO: 84, provided that the amino acidsequence comprising said stretch of amino acid residues binds IL-6R withthe same, about the same, or a higher affinity compared to the aminoacid sequence comprising said stretch of amino acid residues without the2 or 1 amino acid difference, said affinity as measured by surfaceplasmon resonance.

In yet another specific aspect, the amino acid sequence or Nanobody ofthe invention comprises at least a stretch of amino acid residues chosenfrom SEQ ID NO's: 93-94; or a stretch of amino acid residues that has nomore than 2, preferably no more than 1 amino acid difference with one ofSEQ ID NO's: 93-94, provided that the amino acid sequence comprisingsaid stretch of amino acid residues binds IL-6R with the same, about thesame, or a higher affinity compared to the amino acid sequencecomprising said stretch of amino acid residues without the 2 or 1 aminoacid difference, said affinity as measured by surface plasmon resonance.

In yet another specific aspect, the amino acid sequence or Nanobody ofthe invention comprises at least SEQ ID NO: 93; or a stretch of aminoacid residues that has no more than 2, preferably no more than 1 aminoacid difference with SEQ ID NO: 93, provided that the amino acidsequence comprising said stretch of amino acid residues binds IL-6R withthe same, about the same, or a higher affinity compared to the aminoacid sequence comprising said stretch of amino acid residues without the2 or 1 amino acid difference, said affinity as measured by surfaceplasmon resonance.

In yet another specific aspect, the amino acid sequence or Nanobody ofthe invention comprises at least SEQ ID NO: 80; or a stretch of aminoacid residues that has no more than 2, preferably no more than 1 aminoacid difference with SEQ ID NO: 80; and at least a stretch of amino acidresidues chosen from SEQ ID NO's: 84, 89 or 91; or a stretch of aminoacid residues that has no more than 2, preferably no more than 1 aminoacid difference with one of SEQ ID NO's: 84, 89 or 91, provided that theamino acid sequence comprising said stretches of amino acid residuesbinds IL-6R with the same, about the same, or a higher affinity comparedto the amino acid sequence comprising said stretches of amino acidresidues without the 2 or 1 amino acid difference, said affinity asmeasured by surface plasmon resonance.

In yet another specific aspect, the amino acid sequence or Nanobody ofthe invention comprises at least SEQ ID NO: 80; or a stretch of aminoacid residues that has no more than 2, preferably no more than 1 aminoacid difference with SEQ ID NO: 80; and at least SEQ ID NO: 84; or astretch of amino acid residues that has no more than 2, preferably nomore than 1 amino acid difference with SEQ ID NO: 84, provided that theamino acid sequence comprising said stretches of amino acid residuesbinds IL-6R with the same, about the same, or a higher affinity comparedto the amino acid sequence comprising said stretches of amino acidresidues without the 2 or 1 amino acid difference, said affinity asmeasured by surface plasmon resonance.

In yet another specific aspect, the amino acid sequence or Nanobody ofthe invention comprises at least SEQ ID NO: 80; or a stretch of aminoacid residues that has no more than 2, preferably no more than 1 aminoacid difference with SEQ ID NO: 80; and at least a stretch of amino acidresidues chosen from SEQ ID NO's: 93-94; or a stretch of amino acidresidues that has no more than 2, preferably no more than 1 amino aciddifference with one of SEQ ID NO's: 93-94, provided that the amino acidsequence comprising said stretches of amino acid residues binds IL-6Rwith the same, about the same, or a higher affinity compared to theamino acid sequence comprising said stretches of amino acid residueswithout the 1 or 2 amino acid difference, said affinity as measured bysurface plasmon resonance.

in yet another specific aspect, the amino acid sequence or Nanobody ofthe invention comprises at least SEQ ID NO: 80; or a stretch of aminoacid residues that has no more than 2, preferably no more than 1 aminoacid difference with SEQ ID NO: 80; and at least SEQ ID NO: 93; or astretch of amino acid residues that has no more than 2, preferably nomore than 1 amino acid difference with SEQ ID NO: 93, provided that theamino acid sequence comprising said stretches of amino acid residuesbinds IL-6R with the same, about the same, or a higher affinity comparedto the amino acid sequence comprising said stretches of amino acidresidues without the 2 or 1 amino acid difference, said affinity asmeasured by surface plasmon resonance.

In yet another specific aspect, the amino acid sequence or Nanobody ofthe invention comprises at least a stretch of amino acid residues chosenfrom SEQ ID NO's: 84, 89, or 91; or a stretch of amino acid residuesthat has no more than 2, preferably no more than 1 amino acid differencewith one of SEQ ID NO's: 84, 89, or 91; and at least a stretch of aminoacid residues chosen from SEQ ID NO's: 93-94; or a stretch of amino acidresidues that has no more than 2, preferably no more than 1 amino aciddifference with one of SEQ ID NO's: 93-94, provided that the amino acidsequence comprising said stretches of amino acid residues binds IL-6Rwith the same, about the same, or a higher affinity compared to theamino acid sequence comprising said stretches of amino acid residueswithout the 2 or 1 amino acid difference, said affinity as measured bysurface plasmon resonance.

In yet another specific aspect, the amino acid sequence or Nanobody ofthe invention comprises at least a stretch of amino acid residues chosenfrom SEQ ID NO's: 84, 89, or 91; or a stretch of amino acid residuesthat has no more than 2, preferably no more than 1 amino acid differencewith one of SEQ ID NO's: 84, 89, or 91; and at least SEQ ID NO: 93; or astretch of amino acid residues that has no more than 2, preferably nomore than 1 amino acid difference with SEQ ID NO: 93, provided that theamino acid sequence comprising said stretches of amino acid residuesbinds IL-6R with the same, about the same, or a higher affinity comparedto the amino acid sequence comprising said stretches of amino acidresidues without the 2 or 1 amino acid difference, said affinity asmeasured by surface plasmon resonance.

In yet another specific aspect, the amino acid sequence or Nanobody ofthe invention comprises at least SEQ ID NO: 84; or a stretch of aminoacid residues that has no more than 2, preferably no more than 1 aminoacid difference with SEQ ID NO: 84; and at least a stretch of amino acidresidues chosen from SEQ ID NO's: 93-94; or a stretch of amino acidresidues that has no more than 2, preferably no more than 1 amino aciddifference with one of SEQ ID NC's: 93-94, provided that the amino acidsequence comprising said stretches of amino acid residues binds IL-6Rwith the same, about the same, or a higher affinity compared to theamino acid sequence comprising said stretches of amino acid residueswithout the 2 or 1 amino acid difference, said affinity as measured bysurface plasmon resonance.

In yet another specific aspect, the amino acid sequence or Nanobody ofthe invention comprises at least SEQ ID NO: 84; or a stretch of aminoacid residues that has no more than 2, preferably no more than 1 aminoacid difference with SEQ ID NO: 84; and at least SEQ ID NO: 93; or astretch of amino acid residues that has no more than 2, preferably nomore than 1 amino acid difference with SEQ ID NO: 93, provided that theamino add sequence comprising said stretches of amino acid residuesbinds IL-6R with the same, about the same, or a higher affinity comparedto the amino acid sequence comprising said stretches of amino acidresidues without the 2 or 1 amino acid difference, said affinity asmeasured by surface plasmon resonance.

In yet another specific aspect, the amino acid sequence or Nanobody ofthe invention comprises at least SEQ ID NO: 80 and SEQ ID NO: 84.

In yet another specific aspect, the amino acid sequence or Nanobody ofthe invention comprises at least SEQ ID NO: 80 and SEQ ID NO: 93.

In yet another specific aspect, the amino acid sequence or Nanobody ofthe invention comprises at least SEQ ID NO: 84 and SEQ ID NO: 93.

In yet another specific aspect, the amino acid sequence or Nanobody ofthe invention comprises at least SEQ ID NO: 80; or a stretch of aminoacid residues that has no more than 2, preferably no more than 1 aminoacid difference with SEQ ID NO: 80; and at least a stretch of amino acidresidues chosen from SEQ ID NO's: 84, 89, or 91; or a stretch of aminoacid residues that has no more than 2, preferably no more than 1 aminoacid difference with one of SEQ ID NO's: 84, 89, or 91; and at least astretch of amino acid residues chosen from SEQ ID NO's: 93-94; or astretch of amino acid residues that has no more than 2, preferably nomore than 1 amino acid difference with one of SEQ ID NO's: 93-94,provided that the amino acid sequence comprising said stretches of aminoacid residues binds IL-6R with the same, about the same, or a higheraffinity compared to the amino acid sequence comprising said stretchesof amino acid residues without the 2 or 1 amino acid difference, saidaffinity as measured by surface plasmon resonance.

In yet another specific aspect, the amino acid sequence or Nanobody ofthe invention comprises at least SEQ ID NO: 80; or a stretch of aminoacid residues that has no more than 2, preferably no more than 1 aminoacid difference with SEQ ID NO: 80; and at least a stretch of amino acidresidues chosen from SEQ ID NO's: 84, 89, or 91; or a stretch of aminoacid residues that has no more than 2, preferably no more than 1 aminoacid difference with one of SEQ ID NO's: 84, 89, or 91; and at least SEQID NO: 93; or a stretch of amino acid residues that has no more than 2,preferably no more than 1 amino acid difference with SEQ ID NO: 93,provided that the amino acid sequence comprising said stretches of aminoacid residues binds IL-6R with the same, about the same, or a higheraffinity compared to the amino acid sequence comprising said stretchesof amino acid residues without the 2 or 1 amino acid difference, saidaffinity as measured by surface plasmon resonance.

In yet another specific aspect, the amino acid sequence or Nanobody ofthe invention comprises at least SEQ ID NO: 80; or a stretch of aminoacid residues that has no more than 2, preferably no more than 1 aminoacid difference with SEQ ID NO: 80; and at least SEQ ID NO: 84; or astretch of amino acid residues that has no more than 2, preferably nomore than 1 amino acid difference with SEQ ID NO: 84; and at least astretch of amino acid residues chosen from SEQ ID NO's: 93-94; or astretch of amino acid residues that has no more than 2, preferably nomore than 1 amino acid difference with one of SEQ ID NO's: 93-94,provided that the amino acid sequence comprising said stretches of aminoacid residues binds IL-6R with the same, about the same, or a higheraffinity compared to the amino acid sequence comprising said stretchesof amino acid residues without the 2 or 1 amino acid difference, saidaffinity as measured by surface plasmon resonance.

In yet another specific aspect, the amino acid sequence or Nanobody ofthe invention comprises at least SEQ ID NO: 80; or a stretch of aminoacid residues that has no more than 2, preferably no more than 1 aminoacid difference with SEQ ID NO: 80; and at least SEQ ID NO: 84; or astretch of amino acid residues that has no more than 2, preferably nomore than 1 amino acid difference with SEQ ID NO: 84; and at least SEQID NO: 93; or a stretch of amino acid residues that has no more than 2,preferably no more than 1 amino acid difference with SEQ ID NO: 93,provided that the amino acid sequence comprising said stretches of aminoacid residues binds IL-6R with the same, about the same, or a higheraffinity compared to the amino acid sequence comprising said stretchesof amino acid residues without the 2 or 1 amino acid difference, saidaffinity as measured by surface plasmon resonance.

In yet another specific aspect, the amino acid sequence or Nanobody ofthe invention comprises at least SEQ ID NO: 80, SEQ ID NO: 84 and SEQ IDNO: 93.

Preferred combinations of CDR1, CDR2, and CDR3 sequences defined hereinfor the amino acid sequences of the invention are also shown in TableA-1.

In a preferred aspect, amino acid sequences of the invention areselected from the group consisting of:

-   -   a) SEQ ID NO's: 60-69;    -   b) an amino acid sequence that has no more than 2, preferably no        more than 1 amino acid difference in one, two or all of its CDR        sequences with one of SEQ ID NO's: 60-69, provided that the        amino acid sequence with no more than 2, preferably no more than        1 amino acid difference in one, two or all of its CDR sequences        binds IL-6R with the same, about the same, or a higher affinity        compared to the binding by the one of SEQ ID ND's: 60-69, said        affinity as measured by surface plasmon resonance; and    -   c) a sequence that has no more than 2, preferably no more than 1        amino acid difference with one of SEQ ID NO's: 60-69, provided        that the amino acid sequence with no more than 2, preferably no        more than 1 amino acid difference with one of SEQ ID NO's: 60-69        binds IL-611 with the same, about the same, or a higher affinity        compared to the binding by the one of SEQ ID NO's: 60-69, said        affinity as measured by surface plasmon resonance.

In another preferred aspect, the amino acid sequences of the inventionare selected from the group consisting of:

-   -   a) SEQ ID NO's: 65-69;    -   b) an amino acid sequence that has no more than 2, preferably no        more than 1 amino acid difference in one, two or all of its CDR        sequences with one of SEQ ID NO's: 65-69, provided that the        amino acid sequence with no more than 2, preferably no more than        1 amino acid difference in one, two or all of its CDR sequences        binds IL-6R with the same, about the same, or a higher affinity        compared to the binding by the one of SEQ ID NO's: 65-69, said        affinity as measured by surface plasmon resonance; and    -   c) a sequence that has no more than 2, preferably no more than 1        amino acid difference with one of SEQ ID NO's: 65-69, provided        that the amino acid sequence with no more than 2, preferably no        more than 1 amino acid difference with one of SEQ ID NO's: 65-69        binds IL-6R with the same, about the same, or a higher affinity        compared to the binding by the one of SEQ ID NO's: 65-69, said        affinity as measured by surface plasmon resonance.

In yet another preferred aspect, the amino acid sequences of theinvention are selected from the group consisting of:

-   -   a) SEQ ID NO: 66;    -   b) an amino acid sequence that has no more than 2, preferably no        more than 1 amino acid difference in one, two or all of its CDR        sequences with SEQ ID NO: 66, provided that the amino acid        sequence with no more than 2, preferably no more than 1 amino        acid difference in one, two or all of its CDR sequences binds        IL-6R with the same, about the same, or a higher affinity        compared to the binding by SEQ ID NO: 66, said affinity as        measured by surface plasmon resonance; and    -   c) a sequence that has no more than 2, preferably no more than 1        amino acid difference with SEQ ID NO: 66, provided that the        amino acid sequence with no more than 2, preferably no more than        1 amino acid difference binds IL-6R with the same, about the        same, or a higher affinity compared to the binding by SEQ ID NO:        66, said affinity as measured by surface plasmon resonance.

When comparing two stretches of amino acid residues (or two CDRsequences), the term “amino acid difference in one, two or all of itsCDRs” refers to an insertion, deletion or substitution of a single aminoacid residue on a position of a stretch of amino acid residues (or CDRsequence) comprised in the amino acid sequence of the inventionspecified in b), compared to the stretch of amino acid residues (or CDRsequence) comprised in the amino acid sequence of the inventionspecified in a); it being understood that two stretches of amino acidresidues (or CDR sequences) can contain one or maximal two such aminoacid differences.

By “amino acid difference in one, two or all of its CDRs” is meant thatamino acid sequence of the invention may have no more than 2, preferablyno more than 1 amino acid difference in its CDR1 and/or no more than 2,preferably no more than 1 amino acid difference in its CDR2, and/or nomore than 2, preferably no more than 1 amino acid difference in its CDR3compared to CDR1, CDR2 and/or CDR3 in one of the amino acid sequences ofa) (i.e. one of SEQ ID NO's: 60-69); such as no more than 2, preferablyno more than 1 amino acid difference in its CDR1 compared to the CDR1 inone of the amino acid sequences of a) (i.e. one of SEQ ID NO's: 60-69);or no more than 2, preferably no more than 1 amino acid difference inits CDR2 compared to the CDR2 in one of the amino acid sequences of a)(i.e. one of SEQ ID NO's: 60-69); or no more than 2, preferably no morethan 1 amino acid difference in its CDR3 compared to the CDR3 in one ofthe amino acid sequences of a) (i.e. one of SEQ ID NO's: 60-69); or nomore than 2, preferably no more than 1 amino acid difference in its CDR1compared to the CDR1 in one of the amino acid sequences of a) (i.e. oneof SEQ ID NO's: 60-69) and no more than 2, preferably no more than 1amino acid difference in its CDR2 compared to the CDR2 in one of theamino acid sequences of a) (i.e. one of SEQ ID NO's: 60-69); or no morethan 2, preferably no more than 1 amino acid difference in its CDR1compared to the CDR1 in one of the amino acid sequences of a) (i.e. oneof SEQ ID NO's: 60-69) and no more than 2, preferably no more than 1amino acid difference in its CDR3 compared to the CDR3 in one of SEQ IDNO's: 60-69; or no more than 2, preferably no more than 1 amino aciddifference in its CDR2 compared to the CDR2 in one of the amino acidsequences of a) (i.e. one of SEQ ID NO's: 60-69) and no more than 2,preferably no more than 1 amino acid difference in its CDR3 compared tothe CDR3 in one of the amino acid sequences of a) (i.e. one of SEQ IDNO's: 60-69); or no more than 2, preferably no more than 1 amino aciddifference in its CDR1 compared to the CDR1 in one of the amino acidsequences of a) (i.e. one of SEQ ID NO's: 60-69), no more than 2,preferably no more than 1 amino acid difference in its CDR2 compared tothe CDR2 in one of the amino acid sequences of a) (i.e. one of SEQ IDNO's: 60-69) and no more than 2, preferably no more than 1 amino aciddifference in its CDR3 compared to the CDR3 in one of the amino acidsequences of a) (i.e. one of SEQ ID NO's: 60-69).

The “amino acid difference in one, two or all of its CORE” can be anyone or maximal two substitutions, deletions or insertions in one or moreof the CDRs, or any combination thereof, that either improve theproperties of the amino acid sequence of the invention or that at leastdo not detract too much from the desired properties or from the balanceor combination of desired properties of the amino acid sequence of theinvention. In this respect, the resulting amino acid sequence of theinvention should at least bind IL-6R with the same, about the same or ahigher affinity compared to the amino acid sequence comprising the oneor more stretch of amino acid residues without the one or maximal twosubstitutions, deletions or insertions, said affinity as measured bysurface plasmon resonance. The resulting amino acid sequences arepreferably such that they can bind to the specific epitope on the IL-6receptor, with affinity (suitably measured and/or expressed as aK_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), ak_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, asfurther described herein) that is as defined herein. The resulting aminoacid sequences also preferably have a cell based potency and a plasmapotency as defined herein.

In one aspect of the invention, the “amino acid difference in one, twoor all of its CDRs” is an amino acid substitution. The amino acidsubstitution may be any one or maximal two substitutions in one or moreof the CDRs that either improve the properties of the amino acidsequence of the invention or that at least do not detract too much fromthe desired properties or from the balance or combination of desiredproperties of the amino acid sequence of the invention. In this respect,the resulting amino acid sequence of the invention should at least bindIL-6R with the same, about the same or a higher affinity compared to theamino acid sequence comprising the one or more stretches of amino acidresidues without the one or maximal two substitutions, said affinity asmeasured by surface plasmon resonance. The resulting amino acid sequenceare preferably such that they can bind to the specific epitope on theIL-6 receptor, with affinity (suitably measured and/or expressed as aK_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), ak_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, asfurther described herein) that is as defined herein. The resulting aminoacid sequences also preferably have a cell based potency and a plasmapotency as defined herein.

As discussed above, the amino acid substitution in the CDRs may be anypossible substitution such as a “conservative substitution” (as definedherein), it may be driven by certain rules (as defined herein), and/orit may induce improved properties to the resulting amino acid sequences.

The invention also relates to an amino acid sequence that has no morethan 2, preferably no more than 1 amino acid difference with one of (thefull sequence of) SEQ ID NO's: 60-69.

When comparing two amino acid sequences, the term “amino aciddifference” refers to an insertion, deletion or substitution of a singleamino acid residue on a position of the first amino acid sequence,compared to the second amino acid sequence; it being understood that twoamino acid sequences can contain one or maximal two such amino aciddifferences.

The “amino acid difference” can be any one or maximal any twosubstitutions, deletions or insertions in the amino acid sequence, i.e.in one or more of the framework regions or in one or more of the CDRs,or any combination thereof, that either improve the properties of theamino acid sequence of the invention or that at least do not detract toomuch from the desired properties or from the balance or combination ofdesired properties of the amino acid sequence of the invention. In thisrespect, the resulting amino acid sequence of the invention should atleast bind IL-6R with the same, about the same, or a higher affinitycompared to the amino acid sequence without the one or maximal twosubstitutions, deletions or insertions, said affinity as measured bysurface plasmon resonance. The resulting amino acid sequences arepreferably such that they can bind to the specific epitope on the IL-6receptor, with affinity (suitably measured and/or expressed as aK_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), ak_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, asfurther described herein) that is as defined herein. The resulting aminoacid sequences also preferably have a cell based potency and a plasmapotency as defined herein. The skilled person will generally be able todetermine and select suitable substitutions, deletions or insertions, orsuitable combinations thereof, and determining their influence on theproperties of the amino acid sequence thus obtained.

In one aspect of the invention, the “amino acid difference” is an aminoacid substitution. The amino acid substitution may be any one or maximaltwo substitutions in one or more of the framework regions or in one ormore of the CDRs, or any combination thereof, that either improve theproperties of the amino acid sequence of the invention or that at leastdo not detract too much from the desired properties or from the balanceor combination of desired properties of the amino acid sequence of theinvention. In this respect, the resulting amino acid sequence of theinvention should at least bind IL-6R with the same, about the same, or ahigher affinity compared to the amino acid sequence without the one ormaximal two substitutions, said affinity as measured by surface plasmonresonance. The resulting amino acid sequences are preferably such thatthey can bind to the specific epitope on the IL-6 receptor, withaffinity (suitably measured and/or expressed as a K_(D)-value (actual orapparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or ak_(off)-rate, or alternatively as an IC₅₀ value, as further describedherein) that is as defined herein. The resulting amino acid sequencesalso preferably have a cell based potency and a plasma potency asdefined herein. The skilled person will generally be able to determineand select suitable substitutions, and determining their influence onthe properties of the amino acid sequences thus obtained.

As indicated above, the substitutions, insertions or deletions can be inone or more of the framework regions and/or in one or more of the CDR's.As discussed above, the amino acid substitution in one or more of theCDRs can be any substitution such as a “conservative substitution” (asdefined herein), it may be driven by certain rules (as defined herein),and/or it may induce improved properties to the resulting amino acidsequences.

When such substitutions, insertions or deletions are made in one or moreof the framework regions, they may be made at one or more of theHallmark residues (as e.g. defined in WO 08/020,079; Tables A-3 to A-8)and/or at one or more of the other positions in the framework residues,although substitutions, insertions or deletions at the Hallmark residuesare generally less preferred (unless these are suitable humanizingsubstitutions as described herein). By means of non-limiting examples, asubstitution may for example be a conservative substitution (asdescribed herein) and/or an amino acid residue may be replaced byanother amino acid residue that naturally occurs at the same position inanother V_(HH) domain (see WO 08/020,079, Tables A-5 to A-8), althoughthe invention is generally not limited thereto.

Substitutions, insertions or deletions made (preferably) in one or moreof the framework regions may be substitutions for sequence optimizationof the framework regions such as e.g. humanizing substitution. Somepreferred, but non-limiting humanizing substitutions (and suitablecombinations thereof) will become clear to the skilled person based onthe disclosure herein. Potentially useful humanizing substitutions canbe ascertained by comparing the sequence of the framework regions of oneof the amino acid sequence of the invention defined in a) with thecorresponding framework sequence of one or more closely related humanV_(H) sequences, after which one or more of the potentially usefulhumanizing substitutions (or combinations thereof) thus determined canbe introduced into said amino acid sequence of the invention defined ina) (in any manner known per se, as further described herein) and theresulting humanized amino acid sequence can be tested for affinity forIL-6R, for stability, for ease and level of expression, and/or for otherdesired properties defined herein. In this way, by means of a limiteddegree of trial and error, other suitable humanizing substitutions (orsuitable combinations thereof) can be determined by the skilled personbased on the disclosure herein.

Depending on the host organism used to express the amino acid sequence,Nanobody or polypeptide of the invention, such deletions and/orsubstitutions may also be designed in such a way that one or more sitesfor post-translational modification (such as one or more glycosylationsites) are removed, as will be within the ability of the person skilledin the art. Alternatively, substitutions or insertions may be designedso as to introduce one or more sites for attachment of functional groups(as described herein), for example to allow site-specific pegylation(again as described herein).

As can be seen from the data on the V_(HH) entropy and V_(HH)variability given in Tables A-5-A-8 of WO 08/020,079, some amino acidresidues in the framework regions are more conserved than others.Generally, although the invention in its broadest sense is not limitedthereto, any substitutions, deletions or insertions are preferably madeat positions that are less conserved. Also, generally, amino acidsubstitutions are preferred over amino acid deletions or insertions.

The resulting amino acid sequences of the invention or Nanobodies of theinvention should preferably bind to IL-6R with an affinity (suitablymeasured and/or expressed as a K_(D)-value (actual or apparent), aK_(A)-value (actual or apparent), a k_(on)-rate and/or a k_(off)-rate,or alternatively as an IC₅₀ value, as further described herein)preferably such that they:

-   -   bind to hIL-6R with a dissociation constant (K_(D)) of 1 nM to 1        pM moles/litre or less, preferably 500 pM to 1 pM moles/litre or        less, more preferably 100 pM to 1 pM moles/litre or less, or        even more preferably about 50 pM to 1 pM or less;        and/or such that they:    -   bind to cyno IL-6R with a dissociation constant (K_(D)) of 1 nM        to 1 pM moles/litre or less, preferably 500 pM to 1 pM        moles/litre or less, more preferably 100 pM to 1 pM moles/litre        or less, or even more preferably about 50 pM to 1 pM or less;        and/or such that they:    -   bind to hIL-6R with a k_(on)-rate of between 10⁴ M⁻¹s⁻¹ to about        10⁷ M⁻¹s⁻¹, preferably between 10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more        preferably about 10⁶ M⁻¹ s⁻¹ or more;        and/or such that they:    -   bind to cyno IL-6R with a k_(on)-rate of between 10⁴ M⁻¹s⁻¹ to        about 10⁷ M⁻¹s⁻¹, preferably between 10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹,        more preferably about 10⁶ M⁻¹s⁻¹ or more;        and/or such that they:    -   bind to hIL-6R with a k_(off) rate between 10⁻³ s⁻¹        (t_(1/2)=0.69 s) and 10⁻⁶ s⁻¹ (providing a near irreversible        complex with a t_(1/2) of multiple days), preferably between        10⁻¹ s⁻¹ and 10⁻⁶ s⁻¹, more preferably between 10⁻⁵ s⁻¹ and 10⁻⁶        s⁻¹, such as about 10⁻⁵ s⁻¹ or lower;        and/or such that they:    -   bind to cyno IL-6R with a k_(off) rate between 10⁻³ s⁻¹        (t_(1/2)=0.69 s) and 10⁻⁶ s⁻¹ (providing a near irreversible        complex with a t_(1/2) of multiple days), preferably between        10⁻⁴s⁻¹ and 10⁻⁶ s⁻¹, more preferably between 10⁻⁵ s⁻¹ and 10⁻⁶        s⁻¹, such as about 10⁻⁵ s⁻¹ or lower.

Some preferred IC50 values for binding of the amino acid sequences ofthe invention to IL-6R will become clear from the further descriptionand examples herein.

The potency and/or efficacy of the amino acid sequences and Nanobodiesof the invention, and of compositions comprising the same, can be testedusing any suitable in vitro assay, cell-based assay, in vivo assayand/or animal model known per se, or any combination thereof, dependingon the specific disease or disorder involved. Suitable assays and animalmodels will be clear to the skilled person, and for example includeproliferation assays using IL-6-dependent cell lines including TF-1, XG1and 7TD1, collagen induced arthritis model, transplant model of synovialtissue in SCID mice, xenograft models of various human cancers,including lymphoma, myeloma, prostate cancer and renal cell carcinoma,IBD models including TNBS, primate models (such as e.g. described inShinkura et al., 1998, Anticancer Research 18: 1217-1222), non-humanprimate models of arthritic disease (as e.g. described in Vierboom etal., 2008, Drug Discov. Today: Dis Model doi:10.1016/j.ddmod.2008.06.003) as well as the assays and animal models used in theexperimental part below and in the prior art cited herein (Peake et al.,2006, Rheumatology 45: 1485-9; Wahid et al., 2000, Clin. Exp. Immunol.,122: 133-142; Matsuno et al., 1998, Arthritis and rheumatism 41:2014-2021; WO 08/020,079).

For example, in the TF-1 assay as described by Kitamura et al. (1989, J.Cell Physiol. 140: 323), the amino acid sequences of the invention orNanobodies of the invention may have IC50 values (at 100 IU/mL IL-6)between 10 nM and 50 pM, preferably between 5 nM and 50 pM, morepreferably between 1 nM and 50 pM or less, such as about 750 or 500 pMor less. In this TF-1 assay the amino acid sequences of the invention orNanobodies of the invention may have IC50 values (at 5000 IU/mL IL-6)between 50 nM and 1 nM, preferably between 25 nM and 1 nM, morepreferably between 10 nM and 1 nM or less, such as about 8 nM or less.In this TF-1 assay, the amino acid sequences of the invention orNanobodies of the invention may have IC50 values that are at least thesame and preferably better, at least two times, preferably three times,more preferably four times, even more preferably 5 times, 7 times ormore than 7 times better compared to the IC50 value obtained for thereference IgG as defined by SEQ ID NO's: 1 and 2 or the reference Fab asdefined by SEQ ID NO's: 3 and 4 (see Example 1). In this TF-1 assay, theamino acid sequences of the invention or Nanobodies of the invention mayhave IC50 values that are at least the same and preferably better, atleast two times, preferably three times, more preferably four times,even more preferably 5 times, 7 times or more than 7 times bettercompared to the IC50 value obtained for Tocilizumab (MRA).

In a plasma potency assay at EC50 values of IL-6 (e.g. in the presenceof 27.29 ng/mL IL-6 as described in Example 45), the amino acidsequences of the invention or Nanobodies of the invention may have IC50values between 500 pM and 50 pM, preferably between 250 pM and 50 pM,more preferably between 200 pM and 50 pM or less, such as 150 pM orless. In a plasma potency assay at EC95 values of IL-6 (e.g. in thepresence of 885 ng/mL IL-6 as described in Example 45) the amino acidsequences of the invention or Nanobodies of the invention may have IC50values between 1000 pM and 100 pM, preferably between 750 pM and 100 pM,more preferably between 500 pM and 100 pM or less, such as 400 pM orless. In this plasma potency assay, the amino acid sequences of theinvention or Nanobodies of the invention may have IC50 values that areat least the same and preferably better, at least two times, preferablythree times, more preferably four times, even more preferably 5 times, 7times or more than 7 times better compared to the IC50 value obtainedfor the reference IgG as defined by SEQ ID NO's: 1 and 2 or thereference Fab as defined by SEQ ID NO's: 3 and 4 (see Example 1). Inthis plasma potency assay, the amino acid sequences of the invention orNanobodies of the invention may have IC50 values that are at least thesame and preferably better, at least two times, preferably three times,more preferably four times, even more preferably 5 times, 7 times ormore than 7 times better compared to the 1050 value obtained forTocilizumab (MRA).

In an assay for defining binding to membrane IL-6R on CHO cells, theamino acid sequences of the invention or Nanobodies of the invention mayhave IC50 values between 10 nM and 100 pM, preferably between 5 nM and100 pM, more preferably between 2 nM and 10 pM or less, such as 2 nM orless.

As will also be clear from the disclosure herein, it is also within thescope of the invention to use parts or fragments, or combinations of twoor more parts or fragments, of the amino acid sequences or Nanobodies ofthe invention as defined herein, and in particular parts or fragments ofthe amino acid sequences of HQ ID NO's: 60-69. Thus, according to oneembodiment of the invention, the term “amino acid sequence of theinvention” or “Nanobody of the invention” in its broadest sense alsocovers such parts or fragments.

Generally, such parts or fragments of the amino acid sequences orNanobodies of the invention (including analogs thereof) have amino acidsequences in which, compared to the amino acid sequence of thecorresponding full length amino acid sequence or Nanobody of theinvention, one or more of the amino acid residues at the N-terminal end,one or more amino acid residues at the C-terminal end, one or morecontiguous internal amino acid residues, or any combination thereof,have been deleted and/or removed.

The parts or fragments are preferably such that they can bind to thespecific epitope on the IL-6 receptor, with an affinity (suitablymeasured and/or expressed as a K_(D)-value (actual or apparent), aK_(A)-value (actual or apparent), a k_(on)-rate and/or a k_(off)-rate,or alternatively as an IC₅₀ value, as further described herein) that isas defined herein.

In particular, amino acid sequences, Nanobodies, and parts or fragmentsare preferably such that they:

-   -   bind to hIL-6R with a dissociation constant (K_(D)) of 1 nM to 1        pM moles/litre or less, preferably 500 pM to 1 pM moles/litre or        less, more preferably 100 pM to 1 pM moles/litre or less, or        even more preferably about 50 pM to 1 pM or less;        and/or such that they:    -   bind to cyno IL-6R with a dissociation constant (K_(D)) of 1 nM        to 1 pM moles/litre or less, preferably 500 pM to 1 pM        moles/litre or less, more preferably 100 pM to 1 pM moles/litre        or less, or even more preferably about 50 pM to 1 pM or less;        and/or such that they:    -   bind to hIL-6R with a k_(on)-rate of between 10⁴ M⁻¹s⁻¹ to about        10⁷ M⁻¹s⁻¹, preferably between 10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more        preferably about 10⁶ M⁻¹s⁻¹ or more;        and/or such that they:    -   bind to cyno IL-6R with a k_(on)-rate of between 10⁴ M⁻¹s⁻¹ to        about 10⁷ M⁻¹s⁻¹, preferably between 10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹,        more preferably about 10⁶ M⁻¹s⁻¹ or more;        and/or such that they:    -   bind to hIL-6R with a k_(off) rate between 10⁻³ s⁻¹        (t_(1/2)=0.69 s) and 10⁻⁶ s⁻¹ (providing a near irreversible        complex with a t_(1/2) of multiple days), preferably between        10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹, more preferably between 10⁻⁵ s⁻¹ and 10⁻⁶        s⁻¹, such as about 10⁻⁵ s⁻¹ or lower;        and/or such that they:    -   bind to cyno IL-6R with a k_(off) rate between 10⁻³ s⁻¹        (t_(1/2)=0.69 s) and 10⁻⁶ s⁻¹ (providing a near irreversible        complex with a t_(1/2) of multiple days), preferably between        10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹, more preferably between 10⁻⁵ s⁻¹ and 10⁻⁶        s⁻¹, such as about 10⁻⁵ s⁻¹ or lower.

The affinity of the parts or fragments against the IL-6 receptor, can bedetermined in a manner known per se, for example using the assaydescribed herein.

Any part or fragment is preferably such that it comprises at least 10contiguous amino acid residues, preferably at least 20 contiguous aminoacid residues, more preferably at least 30 contiguous amino acidresidues, such as at least 40 contiguous amino acid residues, of theamino acid sequence of the corresponding full length amino acid sequenceor Nanobody of the invention.

Also, any part or fragment is such preferably that it comprises at leastone of CDR1, CDR2 and/or CDR3 or at least part thereof (and inparticular at least CDR3 or at least part thereof). More preferably, anypart or fragment is such that it comprises at least one of the CDR's(and preferably at least CDR3 or part thereof) and at least one otherCDR (i.e. CDR1 or CDR2) or at least part thereof, preferably connectedby suitable framework sequence(s) or at least part thereof. Morepreferably, any part or fragment is such that it comprises at least oneof the CDR's (and preferably at least CDR3 or part thereof) and at leastpart of the two remaining CDR's, again preferably connected by suitableframework sequence(s) or at least part thereof.

According to another particularly preferred, but non-limitingembodiment, such a part or fragment comprises at least CDR3, such asFR3, CDR3 and FR4 of the corresponding full length Nanobody of theinvention, i.e. as for example described in the Internationalapplication WO 03/050531 (Lasters et al.).

As already mentioned above, it is also possible to combine two or moreof such parts or fragments (i.e. from the same or different amino acidsequences or Nanobodies of the invention), i.e. to provide further partsor fragments (as defined herein) of an amino acid sequence or a Nanobodyof the invention. It is for example also possible to combine one or moreparts or fragments of an amino acid sequence or a Nanobody of theinvention with one or more parts or fragments of a human V_(H) domain.

According to one preferred embodiment, the parts or fragments have adegree of sequence identity of at least 50%, preferably at least 60%,more preferably at least 70%, even more preferably at least 80%, such asat least 90%, 95% or 99% or more with one of the amino acid sequences orNanobodies of SEQ ID NO's: 60-59.

The parts and fragments, and nucleic acid sequences encoding the same,can be provided and optionally combined in any manner known per se. Forexample, such parts or fragments can be obtained by inserting a stopcodon in a nucleic acid that encodes a full-sized amino acid sequence orNanobody of the invention, and then expressing the nucleic acid thusobtained in a manner known per se (e.g. as described herein).Alternatively, nucleic acids encoding such parts or fragments can beobtained by suitably restricting a nucleic acid that encodes afull-sized amino acid sequence or Nanobody of the invention or bysynthesizing such a nucleic acid in a manner known per se. Parts orfragments may also be provided using techniques for peptide synthesisknown per se.

The invention further relates to compounds or constructs, that compriseor essentially consist of one or more amino acid sequences or Nanobodiesof the invention, and optionally further comprise one or more othergroups, residues, moieties or binding units, optionally linked via oneor more linkers. In a preferred aspect said one or more other groups,residues, moieties or binding units are amino acid sequences. In anotherpreferred aspect, said one or more linkers are one or more amino acidsequences. Such compounds or constructs are also referred to as“polypeptides of the invention”.

A polypeptide of the invention may comprise an amino acid sequence orNanobody of the invention, which is fused at its amino terminal end, atits carboxy terminal end, or both at its amino terminal end and at itscarboxy terminal end to at least one further amino acid sequence, i.e.so as to provide a fusion protein comprising said amino acid sequence orNanobody of the invention and the one or more further amino acidsequence.

The one or more further amino acid sequence may be any suitable and/ordesired amino acid sequence. The further amino acid sequence may or maynot change, alter or otherwise influence the (biological) properties ofthe amino acid sequence or Nanobody of the invention, and may or may notadd further functionality to the amino acid sequence, Nanobody or thepolypeptide of the invention. Preferably, the further amino acidsequence is such that it confers one or more desired properties orfunctionalities to the amino acid sequence, Nanobody or the polypeptideof the invention.

Example of such amino acid sequences will be clear to the skilledperson, and may generally comprise all amino acid sequences that areused in peptide fusions based on conventional antibodies and fragmentsthereof (including but not limited to ScFv's and single domainantibodies). Reference is for example made to the review by Holliger andHudson, Nature Biotechnology, 23, 9, 1126-1136 (2005).

For example, such an amino acid sequence may be an amino acid sequencethat increases the half-life, the solubility, or the absorption, reducesthe immunogenicity or the toxicity, eliminates or attenuates undesirableside effects, and/or confers other advantageous properties to and/orreduces the undesired properties of the polypeptides of the invention,compared to the amino acid sequence or Nanobody of the invention per se.Some non-limiting examples of such amino acid sequences are serumproteins, such as human serum albumin (see for example WO 00/27435) orhaptenic molecules (for example haptens that are recognized bycirculating antibodies, see for example WO 98/22141).

The further amino acid sequence may also provide a second binding site,which binding site may be directed against any desired protein,polypeptide, antigen, antigenic determinant or epitope (including butnot limited to the same protein, polypeptide, antigen, antigenicdeterminant or epitope against which the amino acid sequence or Nanobodyof the invention is directed, or a different protein, polypeptide,antigen, antigenic determinant or epitope). For example, the furtheramino acid sequence may provide a second binding site that is directedagainst a serum protein (such as, for example, human serum albumin oranother serum protein such as IgG), so as to provide increased half-lifein serum, Such amino acid sequences for example include Nanobodies, aswell as the small peptides and binding proteins described in WO91/01743, WO 01/45746 and WO 02/076489 and the dAb's described in WO03/002609 and WO 04/003019. Reference is also made to Harmsen et al.(2005, Vaccine, 23 (41): 4926-42), as well as to EP 0368684, as well asto WO 08/028,977, WO 08/043,821, WO 08/043,822 and WO 08/068,280 ofAblynx N.V.

Preferred amino acid sequences that may provide the amino acid sequencesor Nanobodies of the invention with increased half-life may be chosenfrom SEQ ID NO's: 97-99.

Such amino acid sequences may in particular be directed against serumalbumin (and more in particular human serum albumin) and/or against IgG(and more in particular human IgG). For example, such amino acidsequences may be amino acid sequences that are directed against (human)serum albumin and amino acid sequences that can bind to amino acidresidues on (human) serum albumin that are not involved in binding ofserum albumin to FcRn (see for example WO 06/0122787) and/or amino acidsequences that are capable of binding to amino acid residues on serumalbumin that do not form part of domain III of serum albumin (see againfor example WO 06/0122787); amino acid sequences that have or canprovide an increased half-life (see for example WO 08/028,977); aminoacid sequences against human serum albumin that are cross-reactive withserum albumin from at least one species of mammal, and in particularwith at least one species of primate (such as, without limitation,monkeys from the genus Macaca (such as, and in particular, cynomolgusmonkeys (Macaca fascicularis) and/or rhesus monkeys (Macaca mulatta))and baboon (Papio ursinus), reference is again made to WO 08/028,977);amino acid sequences that can bind to serum albumin in a pH independentmanner (see for example WO 08/043,821) and/or amino acid sequences thatare conditional binders (see for example WO 08/D43822).

According to another embodiment, the one or more further amino acidsequences may comprise one or more parts, fragments or domains ofconventional 4-chain antibodies (and in particular human antibodies)and/or of heavy chain antibodies. For example, although usually lesspreferred, an amino acid sequence or Nanobody of the invention may belinked to a conventional (preferably human) V_(H) or V_(L) domain or toa natural or synthetic analog of a V_(H) or V_(L) domain, againoptionally via a linker sequence (including but not limited to other(single) domain antibodies, such as the dAb's described by Ward et al.).

Accordingly, in the compound or construct of the invention, said one ormore other groups, residues, moieties or binding units may be chosenfrom the group consisting of domain antibodies, amino acid sequencesthat are suitable for use as a domain antibody, single domainantibodies, amino acid sequences that are suitable for use as a singledomain antibody, “dAb”'s, amino acid sequences that are suitable for useas a dAb, or Nanobodies.

In one specific aspect of the invention, the compound, construct orpolypeptide of the invention comprising at least one amino acid sequenceor Nanobody of the invention may have an increased half-life, comparedto the corresponding amino acid sequence or Nanobody of the invention.Some preferred, but non-limiting examples of such compounds, constructsand polypeptides will become clear to the skilled person based on thefurther disclosure herein, and may be for example compounds, constructsand polypeptides that comprise amino acid sequences, Nanobodies orpolypeptides of the invention that have been chemically modified toincrease the half-life thereof (for example, by means of pegylation); orpolypeptides of the invention that comprise at least one amino acidsequence or Nanobody of the invention that is linked to at least onemoiety (and in particular at least one amino acid sequence) thatincreases the half-life of the Nanobody of the invention. Examples ofcompounds, constructs or polypeptides of the invention that comprisesuch half-life extending moieties or amino acid sequences will becomeclear to the skilled person based on the further disclosure herein; andfor example include, without limitation, polypeptides in which the oneor more amino acid sequences or Nanobodies of the invention are suitablelinked to one or more serum proteins or fragments thereof (such as serumalbumin or suitable fragments thereof) or to one or more binding unitsthat can bind to serum proteins (such as, for example, Nanobodies or(single) domain antibodies that can bind to serum proteins such as serumalbumin, serum immunoglobulins such as IgG, or transferrine);polypeptides in which an amino acid sequence or Nanobody of theinvention is linked to an Fc portion (such as a human Fc) or a suitablepart or fragment thereof; or polypeptides in which the one or more aminoacid sequences or Nanobodies of the invention are suitable linked to oneor more small proteins or peptides that can bind to serum proteins (suchas, without limitation, the proteins and peptides described in WO91/01743, WO 01/45746, WO 02/076489).

The at least one amino acid sequence or Nanobody may also be linked toone or more (preferably human) C_(H)1, C_(H)2 and/or C_(H)3 domains,optionally via a linker sequence. For instance, an amino acid sequenceor Nanobody linked to a suitable C_(H)1 domain could for example beused—together with suitable light chains—to generate antibodyfragments/structures analogous to conventional Fab fragments or F(ab′)₂fragments, but in which one or (in case of an F(ab′)₂ fragment) both ofthe conventional V_(H) domains have been replaced by an amino acidsequence or Nanobody of the invention. Also, two amino acid sequences orNanobodies could be linked to a C_(H)3 domain (optionally via a linker)to provide a construct with increased half-life in vivo.

According to one specific aspect of a polypeptide of the invention, oneor more amino acid sequences or Nanobodies of the invention may belinked (optionally via a suitable linker or hinge region) to one or moreconstant domains (for example, 2 or 3 constant domains that can be usedas part of/to form an Fc portion), to an Fe portion and/or to one ormore antibody parts, fragments or domains that confer one or moreeffector functions to the polypeptide of the invention and/or may conferthe ability to bind to one or more Fe receptors. For example, for thispurpose, and without being limited thereto, the one or more furtheramino acid sequences may comprise one or more C_(H)2 and/or C_(H)3domains of an antibody, such as from a heavy chain antibody (asdescribed herein) and more preferably from a conventional human 4-chainantibody; and/or may form (part of) and Fc region, for example from IgG(e.g. from IgG1, IgG2, IgG3 or IgG4), from IgE or from another human Igsuch as IgA, IgD or IgM. For example, WO 94/04678 describes heavy chainantibodies comprising a Camelid V_(HH) domain or a humanized derivativethereof (i.e. a Nanobody), in which the Camelidae C_(H)2 and/or C_(H)3domain have been replaced by human C_(H)2 and C_(H)3 domains, so as toprovide an immunoglobulin that consists of 2 heavy chains eachcomprising a Nanobody and human C_(H)2 and C_(H)3 domains (but no C_(H)1domain), which immunoglobulin has the effector function provided by theC_(H)2 and C_(H)3 domains and which immunoglobulin can function withoutthe presence of any light chains. Other amino acid sequences that can besuitably linked to the amino acid sequences or Nanobodies of theinvention so as to provide an effector function will be clear to theskilled person, and may be chosen on the basis of the desired effectorfunction(s). Reference is for example made to WO 04/058820, WO 99/42077,WO 02/056910 and WO 05/017148, as well as the review by Holliger andHudson, supra; and to WO 09/068,628). Coupling of an amino acid sequenceor Nanobody of the invention to an Fc portion may also lead to anincreased half-life, compared to the corresponding amino acid sequenceor Nanobody of the invention. For some applications, the use of an Pcportion and/or of constant domains (i.e. C_(H)2 and/or C_(H)3 domains)that confer increased half-life without any biologically significanteffector function may also be suitable or even preferred. Other suitableconstructs comprising one or more amino acid sequences or Nanobodies andone or more constant domains with increased half-life in vivo will beclear to the skilled person, and may for example comprise two amino acidsequences or Nanobodies linked to a C_(H)3 domain, optionally via alinker sequence. Generally, any fusion protein or derivatives withincreased half-life will preferably have a molecular weight of more than50 kD, the cut-off value for renal absorption.

In another specific, but non-limiting, aspect, in order to form apolypeptide of the invention, one or more amino acid sequences of theinvention may be linked (optionally via a suitable linker or hingeregion) to naturally occurring, synthetic or semisynthetic constantdomains (or analogs, variants, mutants, parts or fragments thereof) thathave a reduced (or essentially no) tendency to self-associate intodimers (i.e. compared to constant domains that naturally occur inconventional 4-chain antibodies). Such monomeric (i.e. notself-associating) Fc chain variants, or fragments thereof, will be clearto the skilled person. For example, Helm et al. (1996, J. Biol. Chem.271: 7494), describe monomeric Fc chain variants that can be used in thepolypeptide chains of the invention.

Also, such monomeric Fc chain variants are preferably such that they arestill capable of binding to the complement or the relevant Fcreceptor(s) (depending on the Fc portion from which they are derived),and/or such that they still have some or all of the effector functionsof the Fc portion from which they are derived (or at a reduced levelstill suitable for the intended use), Alternatively, in such apolypeptide chain of the invention, the monomeric Fc chain may be usedto confer increased half-life upon the polypeptide chain, in which casethe monomeric Fc chain may also have no or essentially no effectorfunctions.

Generally, the amino acid sequences or Nanobodies of the invention (orcompounds, constructs or polypeptides comprising the same) withincreased half-life preferably have a half-life that is at least 1.5times, preferably at least 2 times, such as at least 5 times, forexample at least 10 times or more than 20 times, greater than thehalf-life of the corresponding amino acid sequence or Nanobody of theinvention per se. For example, the amino acid sequences, Nanobodies,compounds, constructs or polypeptides of the invention with increasedhalf-life may have a half-life that is increased with more than 1 hours,preferably more than 2 hours, more preferably more than 6 hours, such asmore than 12 hours, or even more than 24, 48 or 72 hours, compared tothe corresponding amino acid sequence or Nanobody of the invention perse.

In a preferred, but non-limiting aspect of the invention, such aminoacid sequences, Nanobodies, compound, constructs or polypeptides of theinvention exhibit a serum half-life in human of at least about 12 hours,preferably at least 24 hours, more preferably at least 48 hours, evenmore preferably at least 72 hours or more. For example, compounds orpolypeptides of the invention may have a half-life of at least 5 days(such as about 5 to 10 days), at preferably at least 9 days (such asabout 9 to 14 days), more preferably at least about 10 days (such asabout 10 to 15 days), or at least about 11 days (such as about 11 to 16days), more preferably at least about 12 days (such as about 12 to 18days or more), or more than 14 days (such as about 14 to 19 days).

The further amino acid sequence may also form a signal sequence orleader sequence that directs secretion of the amino acid sequence,Nanobody or the polypeptide of the invention from a host cell uponsynthesis (for example to provide a pre-, pro- or prepro-form of thepolypeptide of the invention, depending on the host cell used to expressthe polypeptide of the invention).

The further amino acid sequence may also form a sequence or signal thatallows the amino acid sequence, Nanobody or polypeptide of the inventionto be directed towards and/or to penetrate or enter into specificorgans, tissues, cells, or parts or compartments of cells, and/or thatallows the amino acid sequence, Nanobody or polypeptide of the inventionto penetrate or cross a biological barrier such as a cell membrane, acell layer such as a layer of epithelial cells, a tumor including solidtumors, or the blood-brain-barrier. Suitable examples of such amino acidsequences will be clear to the skilled person, and for example include,but are not limited to, the “Peptrans” vectors mentioned above, thesequences described by Cardinale et al. and the amino acid sequences andantibody fragments known per se that can be used to express or producethe Nanobodies and polypeptides of the invention as so-called“intrabodies”, for example as described in WO 94/02610, WO 95/22618,U.S. Pat. No. 7,004,940, WO 03/014960, WO 99/07414; WO 05/01690; EP1512696; and in Cattaneo A. and Biocca S. (1997, intracellularAntibodies: Development and Applications. Landes and Springer-Verlag)and in Kontermann, (2004, Methods 34: 163-170, and the furtherreferences described therein.

According to one preferred, but non-limiting embodiment, the amino acidsequence or Nanobody of the invention comprises at least one furtheramino acid sequence or Nanobody, so as to provide a polypeptide of theinvention that comprises at least two, such as two, three, four, five ormore amino acid sequences or Nanobodies, in which said amino acidsequences or Nanobodies may optionally be linked via one or more linkersequences (as defined herein). Polypeptides of the invention thatcomprise two or more amino acid sequences or Nanobodies, of which atleast one is a amino acid sequence or Nanobody of the invention, willalso be referred to herein as “multivalent” polypeptides of theinvention, and the amino acid sequences or Nanobodies present in suchpolypeptides will also be referred to herein as being in a “multivalentformat”. For example a “bivalent” polypeptide of the invention comprisestwo amino acid sequences and/or Nanobodies, optionally linked via alinker sequence, whereas a “trivalent” polypeptide of the inventioncomprises three amino acid sequences and/or Nanobodies, optionallyfinked via two linker sequences; etc.; in which at least one of theamino acid sequences and/or Nanobodies present in the polypeptide, andup to all of the amino acid sequences and/or Nanobodies present in thepolypeptide, is/are a amino acid sequence and/or Nanobody of theinvention.

In a multivalent polypeptide of the invention, the two or more aminoacid sequences or Nanobodies may be the same or different, and may bedirected against the same antigen or antigenic determinant (for exampleagainst the same part(s) or epitope(s) or against different parts orepitopes) or may alternatively be directed against different antigens orantigenic determinants; or any suitable combination thereof. Forexample, a bivalent polypeptide of the invention may comprise (a) twoidentical amino acid sequences or Nanobodies; (b) a first amino acidsequence or Nanobody directed against a first antigenic determinant of aprotein or antigen and a second amino acid sequence or Nanobody directedagainst the same antigenic determinant of said protein or antigen whichis different from the first amino acid sequence or Nanobody; (c) a firstamino acid sequence or Nanobody directed against a first antigenicdeterminant of a protein or antigen and a second amino acid sequence orNanobody directed against another antigenic determinant of said proteinor antigen; or (d) a first amino acid sequence or Nanobody directedagainst a first protein or antigen and a second amino acid sequence orNanobody directed against a second protein or antigen (i.e. differentfrom said first antigen). Similarly, a trivalent polypeptide of theinvention may, for example and without being limited thereto. comprise(a) three identical amino acid sequences or Nanobodies; (b) twoidentical amino acid sequences or Nanobody against a first antigenicdeterminant of an antigen and a third amino acid sequence or Nanobodydirected against a different antigenic determinant of the same antigen;(c) two identical amino acid sequences or Nanobodies against a firstantigenic determinant of an antigen and a third amino acid sequence orNanobody directed against a second antigen different from said firstantigen; (d) a first amino acid sequence or Nanobody directed against afirst antigenic determinant of a first antigen, a second amino acidsequence or Nanobody directed against a second antigenic determinant ofsaid first antigen and a third amino acid sequence or Nanobody directedagainst a second antigen different from said first antigen; or (e) afirst amino acid sequence or Nanobody directed against a first antigen,a second amino acid sequence or Nanobody directed against a secondantigen different from said first antigen, and a third amino acidsequence or Nanobody directed against a third antigen different fromsaid first and second antigen.

Polypeptides of the invention that contain at least two amino acidsequences and/or Nanobodies, in which at least one amino acid sequenceor Nanobody is directed against a first antigen (i.e. against the IL-6receptor) and at least one amino acid sequence or Nanobody is directedagainst a second antigen (i.e. different from the IL-6 receptor), willalso be referred to as “multispecific” polypeptides of the invention,and the amino acid sequences or Nanobodies present in such polypeptideswill also be referred to herein as being in a “multispecific format”.Thus, for example, a “bispecific” polypeptide of the invention is apolypeptide that comprises at least one amino acid sequence or Nanobodydirected against a first antigen (i.e. the IL-6 receptor) and at leastone further amino acid sequence or Nanobody directed against a secondantigen (i.e. different from the IL-6 receptor), whereas a “trispecific”polypeptide of the invention is a polypeptide that comprises at leastone amino acid sequence or Nanobody directed against a first antigen(i.e. the IL-6 receptor), at least one further amino acid sequence orNanobody directed against a second antigen (i.e. different from the IL-6receptor) and at least one further amino acid sequence or Nanobodydirected against a third antigen (i.e. different from both the IL-6receptor, and the second antigen); etc.

Accordingly, in its simplest form, a bispecific polypeptide of theinvention is a bivalent polypeptide of the invention (as definedherein), comprising a first amino acid sequence or Nanobody directedagainst the IL-6 receptor, and a second amino acid sequence or Nanobodydirected against a second antigen, in which said first and second aminoacid sequence or Nanobody may optionally be linked via a linker sequence(as defined herein); whereas a trispecific polypeptide of the inventionin its simplest form is a trivalent polypeptide of the invention (asdefined herein), comprising a first amino acid sequence or Nanobodydirected against the IL-6 receptor, a second amino acid sequence orNanobody directed against a second antigen and a third amino acidsequence or Nanobody directed against a third antigen, in which saidfirst, second and third amino acid sequence or Nanobody may optionallybe linked via one or more, and in particular one and more in particulartwo, linker sequences.

In a specific aspect, the polypeptide of the invention is a trivalent,bispecific polypeptide. A trivalent, bispecific polypeptide of theinvention in its simplest form may be a trivalent polypeptide of theinvention (as defined herein), comprising two identical amino acidsequences or Nanobodies against the IL-6 receptor and a third amino acidsequence or Nanobody directed against another antigen, in which saidfirst, second and third amino acid sequence or Nanobody may optionallybe linked via one or more, and in particular one and more in particulartwo, linker sequences.

In another specific aspect, the polypeptide of the invention is abispecific polypeptide. A bispecific polypeptide of the invention in itssimplest form may be a bivalent polypeptide of the invention (as definedherein), comprising a first amino acid sequence or Nanobody against theIL-6 receptor and a second amino acid sequence or Nanobody directedagainst another antigen, in which said first and second amino acidsequence or Nanobody may optionally be linked via a linker sequence.

In a preferred, but non-limiting, example, the multispecific polypeptideof the invention comprises at least one amino acid sequence or Nanobodyof the invention and at least one Nanobody that provides for anincreased half-life. Some preferred, but non-limiting examples of suchNanobodies include Nanobodies directed against serum proteins, such ashuman serum albumin, thyroxine-binding protein, (human) transferrin,fibrinogen, an immunoglobulin such as IgG, IgE or IgM, or one of theother serum proteins listed in WO 04/003019.

For example, for experiments in mice, Nanobodies against mouse serumalbumin (MSA) can be used, whereas for pharmaceutical use, Nanobodiesagainst human serum albumin can be used.

Another embodiment of the present invention is a polypeptide constructas described above wherein said at least one (human) serum protein isany of (human) serum albumin, (human) serum immunoglobulins, (human)thyroxine-binding protein, (human) transferrin, (human) fibrinogen, etc.

Accordingly, in a specific aspect, the polypeptide of the invention is atrivalent, bispecific polypeptide, comprising two identical amino acidsequences or Nanobodies against the IL-6 receptor and a third amino acidsequence or Nanobody directed against (human) serum albumin, in whichsaid first, second and third amino acid sequence or Nanobody mayoptionally be linked via one or more, and in particular one and more, inparticular two, linker sequences.

In another specific aspect, the polypeptide of the invention is abispecific polypeptide comprising a first amino acid sequence orNanobody against the IL-6 receptor and a second amino acid sequence orNanobody directed against (human) serum albumin, in which said first andsecond amino acid sequence or Nanobody may optionally be linked via alinker sequence.

According to a specific, but non-limiting aspect of the invention, thepolypeptides of the invention contain, besides the one or more aminoacid sequences or Nanobodies of the invention, at least one Nanobodyagainst human serum albumin, Although these Nanobodies against humanserum albumin may be as generally described in the applications byAblynx N.V. cited above (see for example WO4/062551), according to aparticularly preferred, but non-limiting embodiment, said Nanobodyagainst human serum albumin essentially consists of an amino acidsequence selected from SEQ ID NO's: 97-99.

Some preferred, but non-limiting examples of polypeptides of theinvention that comprise at least one amino acid sequence or Nanobodyagainst IL-6R and at least one amino acid sequence or Nanobody thatprovides for increased half-life are:

-   -   a) SEQ ID NO's: 70-72;    -   b) a polypeptide sequence that has no more than 2, preferably no        more than 1 amino acid difference in one, two or all of its CDRs        of the invention with one of SEQ ID NO's: 70-72, provided that        the polypeptide sequence with no more than 2, preferably no more        than 1 amino acid difference in one, two or all of its CDRs of        the invention binds IL-6R with the same, about the same, or a        higher affinity compared to the binding by the one of SEQ ID        NO's: 70-72, said affinity as measured by surface plasmon        resonance; and    -   c) a polypeptide sequence that has no more than 2, preferably no        more than 1 amino acid difference with one of SEQ ID NO's:        70-72, provided that the amino acid sequence with no more than        2, preferably no more than 1 amino acid difference with one of        SEQ ID NO's: 70-72 binds IL-6R with the same, about the same, or        a higher affinity compared to the binding by the one of SEQ ID        NO's: 70-72, said affinity as measured by surface plasmon        resonance.

Some preferred, but non-limiting examples of trivalent bispecificpolypeptides of the invention are:

-   -   a) SEQ ID NO's: 71-72;    -   b) a polypeptide sequence that has no more than 2, preferably no        more than 1 amino acid difference in one, two or all of its CDRs        of the invention with one of SEQ ID NO's: 71-72, provided that        the polypeptide sequence with no more than 2, preferably no more        than 1 amino acid difference in one, two or all of its CDRs of        the invention binds IL-6R with the same, about the same, or a        higher affinity compared to the binding by the one of SEQ ID        NO's: 71-72, said affinity as measured by surface plasmon        resonance; and    -   c) a polypeptide sequence that has no more than 2, preferably no        more than 1 amino acid difference with one of SEQ ID NO's:        71-72, provided that the amino acid sequence with no more than        2, preferably no more than 1 amino acid difference with one of        SEQ ID NO's: 71-72 binds IL-6R with the same, about the same, or        a higher affinity compared to the binding by the one of SEQ ID        NO's: 71-72, said affinity as measured by surface plasmon        resonance.

Some preferred, but non-limiting examples of bispecific polypeptides ofthe invention that comprise an amino acid sequence or Nanobody againstIL-6R and an amino acid sequence or Nanobody that provides for increasedhalf-life are:

-   -   a) SEQ ID NO: 70;    -   b) a polypeptide sequence that has no more than 2, preferably no        more than 1 amino acid difference in one, two or all of its CDRs        of the invention with SEQ ID NO: 70, provided that the        polypeptide sequence with no more than 2, preferably no more        than 1 amino acid difference in one, two or all of its CDRs of        the invention binds IL-6R with the same, about the same, or a        higher affinity compared to the binding by SEQ ID NO: 70, said        affinity as measured by surface plasmon resonance; and    -   c) a polypeptide sequence that has no more than 2, preferably no        more than 1 amino acid difference with SEQ ID NO: 70, provided        that the amino acid sequence with no more than 2, preferably no        more than 1 amino acid difference with SEQ ID NO: 70 binds IL-6R        with the same, about the same, or a higher affinity compared to        the binding by SEQ ID NO: 70, said affinity as measured by        surface plasmon resonance.

When comparing two stretches of amino acid residues (or two CDRsequences), the term “amino acid difference in one, two or all of itsCDRs of the invention” refers to an insertion, deletion or substitutionof a single amino acid residue on a position of a stretch of amino acidresidues (or CDR sequence) of the invention comprised in the polypeptideof the invention specified in b) compared to the stretch of amino acidresidues (or CDR sequence) of the invention comprised in the polypeptideof the invention specified in a); it being understood that two stretchesof amino acid residues (or CDR sequences) of the invention can containone or maximal two such amino acid differences.

By “amino acid difference in one, two or all of its CDRs of theinvention” is meant that the amino acid sequence or Nanobody of theinvention comprised in the polypeptide of the invention may have no morethan 2, preferably no more than 1 amino acid difference in its CDR1and/or no more than 2, preferably no more than 1 amino acid differencein its CDR2, and/or no more than 2, preferably no more than 1 amino aciddifference in its CDR3 (i.e. in CDR1, CDR2 and/or CDR3 that form theantigen binding site for binding by the compound or polypeptide of theinvention to the specific eptiope on IL-6R) compared to CDR1, CDR2and/or CDR3 in the amino acid sequence or Nanobody of the inventioncomprised in one of the polypeptides of a) (i.e. one of SEQ ID NO's:60-69); such as no more than 2, preferably no more than 1 amino aciddifference in its CDR1 (i.e. CDR1 that forms the antigen binding sitefor binding by the compound or polypeptide of the invention to thespecific eptiope on IL-6R) compared to CDR1 in the amino acid sequenceor Nanobody of the invention comprised in one of the polypeptides of a)(i.e. CDR1 in one of SEQ ID NO's: 60-69); or no more than 2, preferablyno more than 1 amino acid difference in its CDR2 (i.e. CDR2 that formthe antigen binding site for binding by the compound or polypeptide ofthe invention to the specific eptiope on IL-6R) compared to CDR2 in theamino acid sequence or Nanobody of the invention comprised in one of thepolypeptides of a) (i.e. CDR2 in one of SEQ ID NO's: 60-69); or no morethan 2, preferably no more than 1 amino acid difference in its CDR3(i.e. CDR3 that form the antigen binding site for binding by thecompound or polypeptide of the invention to the specific eptiope onIL-6R) compared to CDR3 in the amino acid sequence or Nanobody of theinvention comprised in one of the polypeptides of a) (i.e. CDR3 in oneof SEQ ID NO's: 60-69); no more than 2, preferably no more than 1 aminoacid difference in its CDR1 (i.e. CDR1 that form the antigen bindingsite for binding by the compound or polypeptide of the invention to thespecific eptiope on IL-6R) compared to CDR1 in the amino acid sequenceor Nanobody of the invention comprised in one of the polypeptides of a)(i.e. CDR1 in one of SEQ ID NO's: 60-69) and no more than 2, preferablyno more than 1 amino acid difference in its CDR2 (i.e. CDR2 that formthe antigen binding site for binding by the compound or polypeptide ofthe invention to the specific eptiope on IL-6R) comprised compared toCDR2 in the amino acid sequence or Nanobody of the invention comprisedin one of the polypeptides of a) (i.e. CDR2 in one of SEQ ID NO's:60-69); or no more than 2, preferably no more than 1 amino aciddifference in its CDR1 (i.e. CDR1 that form the antigen binding site forbinding by the compound or polypeptide of the invention to the specificeptiope on IL-6R) compared to CDR1 in the amino acid sequence orNanobody of the invention comprised in one of the polypeptides of a)(i.e. CDR1 in one of SEQ ID ND's: 60-69) and no more than 2, preferablyno more than 1 amino acid difference in its CDR3 (i.e. CDR3 that formthe antigen binding site for binding by the compound or polypeptide ofthe invention to the specific eptiope on IL-6R) compared to CDR3 in theamino acid sequence or Nanobody of the invention comprised in one of thepolypeptides of a) (i.e. CDR3 in one of SEQ ID NO's: 60-69); or no morethan 2, preferably no more than 1 amino acid difference in its CDR2(i.e. CDR2 that form the antigen binding site for binding by thecompound or polypeptide of the invention to the specific eptiope onIL-6R) compared to CDR2 in the amino acid sequence or Nanobody of theinvention comprised in one of the polypeptides of a) (i.e. CDR2 in oneof SEQ ID NO's: 60-69) and no more than 2, preferably no more than 1amino acid difference in its CDR3 (i.e. CDR3 that form the antigenbinding site for binding by the compound or polypeptide of the inventionto the specific eptiope on IL-6R) compared to CDR3 in the amino acidsequence or Nanobody of the invention comprised in one of thepolypeptides of a) (i.e. CDR3 in one of SEQ ID NO's: 60-69); no morethan 2, preferably no more than 1 amino acid difference in its CDR1(i.e. CDR1 that form the antigen binding site for binding by thecompound or polypeptide of the invention to the specific eptiope onIL-6R) compared to CDR1 in the amino acid sequence or Nanobody of theinvention comprised in one of the polypeptides of a) (i.e. CDR1 in oneof SEQ ID NO's: 60-69) and no more than 2, preferably no more than 1amino acid difference in its CDR2 (i.e. CDR2 that form the antigenbinding site for binding by the compound or polypeptide of the inventionto the specific eptiope on IL-6R) compared to CDR2 in the amino acidsequence or Nanobody of the invention comprised in one of thepolypeptides of a) (i.e. CDR2 in one of SEQ ID NO's: 60-69) and no morethan 2, preferably no more than 1 amino acid difference in its CDR3(i.e. CDR3 that form the antigen binding site for binding by thecompound or polypeptide of the invention to the specific eptiope onIL-6R) compared to CDR3 in the amino acid sequence or Nanobody of theinvention comprised in one of the polypeptides of a) (i.e. CDR3 in oneof SEQ ID NO's: 60-69).

The “amino acid difference in one, two or all of its CDRs” can be anyone or maximal any two substitutions, deletions or insertions in one ormore of the CDRs of the invention, or any combination thereof, thateither improve the properties of the compound or polypeptide of theinvention or that at least do not detract too much from the desiredproperties or from the balance or combination of desired properties ofthe compound or polypeptide of the invention. In this respect, theresulting compound or polypeptide of the invention should at least bindIL-6R with the same, about the same or a higher affinity compared to thecompound or polypeptide comprising the one or more CDRs of the inventionwithout the one or maximal two substitutions, deletions or insertions,said affinity as measured by surface plasmon resonance. The resultingcompounds or polypeptides are preferably such that they can bind to thespecific epitope on the IL-6 receptor, with affinity (suitably measuredand/or expressed as a K_(D)-value (actual or apparent), a K_(A)-value(actual or apparent), a k_(on)-rate and/or a k_(off)-rate, oralternatively as an IC₅₀ value, as further described herein) that is asdefined herein. The resulting compounds or polypeptides also preferablyhave a cell based potency and a plasma potency as defined herein.

In one aspect of the invention, the “amino acid difference in one, twoor all of its CDRs” is an amino acid substitution. The amino acidsubstitution may be any one or maximal any two substitutions in one ormore CDRs of the invention that either improve the properties of thecompound or polypeptide of the invention or that at least do not detracttoo much from the desired properties or from the balance or combinationof desired properties of the compound or construct of the invention. Inthis respect, the resulting compound or polypeptide of the inventionshould at least bind IL-6R with the same, about the same or a higheraffinity compared to the compound or construct comprising the one ormore CDRs of the invention without the one or maximal two substitutions,said affinity as measured by surface plasmon resonance. The resultingcompounds or polypeptides are preferably such that they can bind to thespecific epitope on the IL-6 receptor, with affinity (suitably measuredand/or expressed as a K_(D)-value (actual or apparent), a K_(A)-value(actual or apparent), a k_(on)-rate and/or a k_(off)-rate, oralternatively as an IC₅₀ value, as further described herein) that is asdefined herein. The resulting compounds or polypeptides also preferablyhave a cell based potency and a plasma potency as defined herein. Theskilled person will generally be able to determine and select suitablesubstitutions, based on the disclosure herein and optionally after alimited degree of routine experimentation, which may for example involveintroducing a limited number of possible substitutions and determiningtheir influence on the properties of the compounds or polypeptides thusobtained.

The amino acid substitution in one or more of the CDRs of the inventionmay be any possible substitution such as a “conservative substitution”(as defined herein), it may be driven by certain rules (as definedherein), and/or it may induce improved properties to the resultingcompounds or polypeptides (as is further defined herein).

The invention also relates to a compound or polypeptide that has no morethan 2, preferably no more than 1 amino acid difference with one of (thefull sequence of) SEQ ID NO's: 70-72.

When comparing two compounds or polypeptides, the term “amino aciddifference” refers to an insertion, deletion or substitution of a singleamino acid residue on a position of the first compound or polypeptide,compared to the second compound or polypeptide; it being understood thattwo compounds or polypeptides can contain one or maximal two such aminoacid differences.

The “amino acid difference” can be any one or maximal two substitutions,deletions or insertions in the compound or polypeptide, i.e. in one ormore of the framework regions or in one or more of the CDRs (which maybe in a CDR of the invention (i.e. present in an amino acid sequence orNanobody of the invention) or in another CDR (i.e. present in SEQ ID NO:98)), in a linker sequence, or any combination thereof, that eitherimprove the properties of the compound or polypeptide of the inventionor that at least do not detract too much from the desired properties orfrom the balance or combination of desired properties of the compound orpolypeptide of the invention. In this respect, the resulting compound orpolypeptide of the invention should at least bind IL-6R with the same,about the same, or a higher affinity compared to the compound orpolypeptide without the one or maximal two substitutions, deletions orinsertions, said affinity as measured by surface plasmon resonance. Theresulting compounds or polypeptides are preferably such that they canbind to the specific epitope on the IL-6 receptor, with affinity(suitably measured and/or expressed as a K_(D)-value (actual orapparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or ak_(off)-rate, or alternatively as an IC₅₀ value, as further describedherein) that is as defined herein. The resulting compounds orpolypeptides also preferably have a cell based potency and a plasmapotency as defined herein.

In one aspect of the invention, the “amino acid difference” is an aminoacid substitution. The amino acid substitution may be any one or maximalany two substitutions in the framework regions, in one or more of theCDRs (which may be in a CDR of the invention (i.e. present in an aminoacid sequence or Nanobody of the invention) or in another CDR (i.e.present in SEQ ID NO: 98)), in a linker sequence, or any combinationthereof, that either improve the properties of the compound orpolypeptide of the invention or that at least do not detract too muchfrom the desired properties or from the balance or combination ofdesired properties of the compound or polypeptide of the invention. Inthis respect, the resulting compound or polypeptide of the inventionshould at least bind IL-6R with the same, about the same or a higheraffinity compared to the compound or polypeptide without the one ormaximal two substitutions, said affinity as measured by surface plasmonresonance. The resulting compounds or polypeptides are preferably suchthat they can bind to the specific epitope on the IL-6 receptor, withaffinity (suitably measured and/or expressed as a K_(D)-value (actual orapparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or ak_(off)-rate, or alternatively as an IC₅₀ value, as further describedherein) that is as defined herein. The resulting compounds orpolypeptides also preferably have a cell based potency and a plasmapotency as defined herein.

As indicated above, the substitutions, insertions or deletions can be inone or more of the framework regions, in one or more of the CDR's,and/or in one or more of the linker sequences. The substitutions,insertions or deletions in the CDR's may be any possible substitutions,insertions or deletions such as “conservative substitution” (as definedherein), it may be driven by certain rules (as defined herein), and/orit may induce improved properties to the resulting compounds orpolypeptides.

When such substitutions, insertions or deletions are made in one or moreof the framework regions, they may be any possible substitutions,insertions or deletions. They can be made at one or more of the Hallmarkresidues (as e.g. defined in WO 08/020,079; Tables A-3 to A-8) and/or atone or more of the other positions in the framework residues, althoughsubstitutions, insertions or deletions at the Hallmark residues aregenerally less preferred (unless these are suitable humanizingsubstitutions as described herein). By means of non-limiting examples, asubstitution may for example be a conservative substitution (asdescribed herein) and/or an amino acid residue may be replaced byanother amino acid residue that naturally occurs at the same position inanother V_(HH) domain (see WO 08/020,079, Tables A-5 to A-8), althoughthe invention is generally not limited thereto.

Substitutions, insertions or deletions made (preferably) in one or moreof the framework regions may be sequence optimizing substitutions suchas e.g. humanizing substitution. Some preferred, but non-limitinghumanizing substitutions (and suitable combinations thereof) will becomeclear to the skilled person based on the disclosure herein. Potentiallyuseful humanizing substitutions can be ascertained by comparing thesequence of the framework regions of one of the amino acid sequence orNanobodies comprised in one of the polypeptides of the invention definedin a) with the corresponding framework sequence of one or more closelyrelated human V_(H) sequences, after which one or more of thepotentially useful humanizing substitutions (or combinations thereof)thus determined can be introduced into said amino acid sequence orNanobody comprised in one of the polypeptides of the invention definedin a) (in any manner known per se, as further described herein) and theresulting humanized polypeptide can be tested for affinity for IL-6R,for stability, for ease and level of expression, and/or for otherdesired properties defined herein. In this way, by means of a limiteddegree of trial and error, other suitable humanizing substitutions (orsuitable combinations thereof) can be determined by the skilled personbased on the disclosure herein.

Depending on the host organism used to express the compound orpolypeptide of the invention, deletions and/or substitutions may also bedesigned in such a way that one or more sites for post-translationalmodification (such as one or more glycosylation sites) are removed, aswill be within the ability of the person skilled in the art.Alternatively, substitutions or insertions may be designed so as tointroduce one or more sites for attachment of functional groups (asdescribed herein), for example to allow site-specific pegylation (againas described herein).

As can be seen from the data on the V_(HH) entropy and V_(HH)variability given in Tables A-5-A-8 of WO 08/020,079, some amino acidresidues in the framework regions are more conserved than others,Generally, although the invention in its broadest sense is not limitedthereto, any substitutions, deletions or insertions are preferably madeat positions that are less conserved. Also, generally, amino acidsubstitutions are preferred over amino acid deletions or insertions.

The resulting compounds of the invention or polypeptides of theinvention should preferably bind to IL-6R with an affinity (suitablymeasured and/or expressed as a K_(D)-value (actual or apparent), aK_(A)-value (actual or apparent), a k_(on)-rate and/or a k_(off)-rate,or alternatively as an IC₅₀ value, as further described herein)preferably such that they:

-   -   bind to hIL-6R with a dissociation constant (K_(D)) of 1 nM to 1        pM moles/litre or less, preferably 500 pM to 1 pM moles/litre or        less, more preferably 100 pM to 1 pM moles/litre or less, or        even more preferably about 50 pM to 1 pM or less;        and/or such that they:    -   bind to cyno IL-6R with a dissociation constant (K_(D)) of 1 nM        to 1 pM moles/litre or less, preferably 500 pM to 1 pM        moles/litre or less, more preferably 100 pM to 1 pM moles/litre        or less, or even more preferably about 50 pM to 1 pM or less;        and/or such that they:    -   bind to hIL-6R with a k_(on)-rate of between 10⁴ M⁻¹s⁻¹ to about        10⁷ M⁻¹s⁻¹, preferably between 10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more        preferably about 10⁶ M⁻¹s⁻¹ or more;        and/or such that they:    -   bind to cyno IL-6R with a k_(on)-rate of between 10⁴ M⁻¹s⁻¹ to        about 10⁷ M⁻¹ s⁻¹, preferably between 10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹,        more preferably about 10⁶ M⁻¹s⁻¹ or more;        and/or such that they:    -   bind to hIL-6R with a k_(off) rate between 10⁻³ s⁻¹        (t_(1/2)=0.69 s) and 10⁻⁶ s⁻¹ (providing a near irreversible        complex with a t_(1/2) of multiple days), preferably between        10⁻⁴ s⁻¹ and 10⁻⁶s⁻¹, more preferably between 10⁻⁵ s⁻¹ and 10⁻⁶        s⁻¹, such as about 10⁻⁵ s⁻¹ or lower;        and/or such that they:    -   bind to cyno IL-6R with a k_(off) rate between 10⁻³ s⁻¹        (t_(1/2)=0.69 s) and 10⁻⁶ s⁻¹ (providing a near irreversible        complex with a t_(1/2) of multiple days), preferably between        10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹, more preferably between 10⁻⁵ and 10⁻⁶        s⁻¹, such as about 10⁻⁵ s⁻¹ or lower.

Some preferred IC₅₀ values for binding of the compounds or polypeptidesof the invention to IL-6R will become clear from the further descriptionand examples herein.

The potency and/or efficacy of the polypeptides and compounds of theinvention, and of compositions comprising the same, can be tested usingany suitable in vitro assay, cell-based assay, in vivo assay and/oranimal model known per se, or any combination thereof, depending on thespecific disease or disorder involved. Suitable assays and animal modelswill be clear to the skilled person, and for example includeproliferation assays using IL-6-dependent cell lines including TF-1, XG1and 7TD1, collagen induced arthritis model, transplant model of synovialtissue in SCID mice, xenograft models of various human cancers,including lymphoma, myeloma, prostate cancer and renal cell carcinoma,IBD models including TNBS, primate models (such as e.g. described inShinkura et al. 1998, Anticancer Research 18: 1217-1222), non-humanprimate models of arthritic disease (as e.g. described in Vierboom etal., 2008, Drug Discov. Today: Dis Model doi:10.1016/j.ddmod.2008.06.003) as well as the assays and animal models used in theexperimental part below and in the prior art cited herein (Peale et al.,2006, Rheumatology 45: 1485-9; Wahid et al., 2000, Clin. Exp. Immunol.,122: 133-142; Matsuno et al., 1998, Arthritis and rheumatism 41:2014-2021; WO 08/020,079).

For example, in the TF-1 assay as described by Kitamura et al. (1989, J.Cell Physiol. 140: 323), the compounds of the invention or polypeptidesof the invention may have IC50 values (at 1001 U/mL IL-6) between 10 nMand 50 pM, preferably between 5 nM and 50 pM, more preferably between 1nM and 50 pM or less, such as about 750 or 500 pM or less. In this TF-1assay the compounds of the invention or polypeptides of the inventionmay have IC50 values (at 5000 IU/mL IL-6) between 50 nM and 1 nM,preferably between 25 nM and 1 nM, more preferably between 10 nM and 1nM or less, such as about 8 nM or less. In this TF-1 assay, thecompounds of the invention or polypeptides of the invention may haveIC50 values that are at least the same and preferably better, at leasttwo times, preferably three times, more preferably four times, even morepreferably 5 times, 7 times or more than 7 times better compared to theIC50 value obtained for the reference IgG as defined by SEQ ID NO's: 1and 2 or the reference Fab as defined by SEQ ID NO's: 3 and 4 (seeExample 1). In this TF-1 assay, the compounds of the invention orpolypeptides of the invention may have IC50 values that are at least thesame and preferably better, at least two times, preferably three times,more preferably four times, even more preferably 5 times, 7 times ormore than 7 times better compared to the IC50 value obtained forTocilizumab (MRA).

in a plasma potency assay at EC50 values of IL-6 (e.g. in the presenceof 27.29 ng/mL IL-6 as described in Example 45), the compounds of theinvention or polypeptides of the invention may have IC50 values between500 pM and 50 pM, preferably between 250 pM and 50 pM, more preferablybetween 200 pM and 50 pM or less, such as 150 pM or less. In a plasmapotency assay at EC95 values of IL-6 (e.g. in the presence of 885 ng/mLIL-6 as described in Example 45) the compounds of the invention orpolypeptides of the invention may have IC50 values between 1000 pM and100 pM, preferably between 750 pM and 100 pM, more preferably between500 pM and 100 pM or less, such as 400 pM or less. In this plasmapotency assay, the compounds of the invention or polypeptides of theinvention may have IC50 values that are at least the same and preferablybetter, at least two times, preferably three times, more preferably fourtimes, even more preferably 5 times, 7 times or more than 7 times bettercompared to the IC50 value obtained for the reference IgG as defined bySEQ ID NO's: 1 and 2 or the reference Fab as defined by SEQ ID NO's: 3and 4 (see Example 1). In this plasma potency assay, the compounds ofthe invention or polypeptides of the invention may have IC50 values thatare the at least the same and preferably better, at least two times,preferably three times, more preferably four times, even more preferably5 times, 7 times or more than 7 times better compared to the IC50 valueobtained for Tocilizumab (MRA).

In an assay for defining binding to membrane IL-6R on CHO cells, thecompounds of the invention or polypeptides of the invention may haveIC50 values between 10 nM and 100 pM, preferably between 5 nM and 100pM, more preferably between 2 nM and 10 pM or less, such as 2 nM orless.

In a preferred aspect, the compound or polypeptide of the invention hasor essentially consists of the amino acid sequence of SEQ ID NO: 70. Inanother preferred aspect, the compound or polypeptide of the inventionhas or essentially consists the amino acid sequence of SEQ ID NO: 71.Polypeptides with these amino acid sequences show improved propertiessuch as e.g. improved binding and/or affinity, improved avidity,improved efficacy and potency, and/or an increased selectivity, inaddition to their capacity to partially or totally block the IL-6/IL-6Rinteraction, and/or inhibit signalization through, IL-6, IL-6R and/orthe IL-6/IL-6R complex.

The invention also relates to a monovalent construct (also referred toas “monovalent construct of the invention”), comprising or essentiallyconsisting of one amino acid sequence or Nanobody of the invention.Preferred monovalent constructs of the invention comprise or essentiallyconsist of SEQ ID NO's: 60-69, such as SEQ ID NO's: 65-69, such as e.g.SEQ ID NO: 66. Such a monovalent constructs, as well as the amino acidsequences and Nanobodies of the invention can be used for thepreparation of a compound or polypeptide of the invention, such as e.g.the multivalent and/or multispecific compounds or polypeptides of theinvention.

Accordingly, the present invention also relates to the use of an aminoacid sequence, Nanobody or monovalent construct of the invention for thepreparation of a compound, construct or polypeptide of the invention.The invention further relates to a method for the preparation of acompound, construct or polypeptide of the invention, comprising thelinking of an amino acid sequence, Nanobody or monovalent construct ofthe invention to one or more other groups, residues, moieties or bindingunits. Such a method may comprise the linking of an amino acid sequence,Nanobody or monovalent construct of the invention to one or more othergroups, residues, moieties or binding units via one or more linkers.

In a preferred aspect the one or more other groups, residues, moietiesor binding units are binding units, such as amino acid sequences orNanobodies. Accordingly, the present invention also relates to the useof an amino acid sequence, Nanobody or monovalent construct of theinvention for the preparation of a multivalent and/or multispecificcompound, construct or polypeptide of the invention. The inventionfurther relates to a method for the preparation of a multivalent and/ormultispecific compound, construct or polypeptide of the invention,comprising the linking of an amino acid sequence, Nanobody or monovalentconstruct of the invention to one or more other binding units, such asamino acid sequences or Nanobodies. Such a method may comprise thelinking of an amino acid sequence, Nanobody or monovalent construct ofthe invention to one or more binding units via one or more linkers.

In a specific aspect, the present invention also relates to the use of amonovalent construct comprising or essentially consisting of one of SEQID NO's: 60-69 (preferably SEQ ID NO's: 65-69, more preferably SEQ IDNO: 66) for the preparation of a multivalent and/or multispecificcompound, construct or polypeptide of the invention. The inventionfurther relates to a method for the preparation of a multivalent and/ormultispecific compound, construct or polypeptide of the invention,comprising the linking of a monovalent construct comprising oressentially consisting of one of SEQ ID NO's: 60-69 (preferably SEQ. IDNO's: 65-69, more preferably SEQ ID NO: 66) to one or more other bindingunits, such as amino acid sequences or Nanobodies. Such a method maycomprise the linking of a monovalent construct comprising or essentiallyconsisting of one of SEQ ID NO's: 60-69 (preferably SEQ ID NO's: 65-69,more preferably SEQ ID NO: 66) to one or more binding units via one ormore linkers.

In another specific aspect, the present invention relates to the use ofa monovalent construct comprising or essentially consisting of one ofSEQ ID NO's: 60-69 (preferably SEQ ID NO's: 65-69, more preferably SEQID NO: 66) for the preparation of a multivalent and/or multispecificcompound, construct or polypeptide comprising or essentially consistingof SEQ ID NO's: 70-72 (preferably SEQ ID NO's: 70-71, more preferablySEQ ID NO: 70 or SEQ ID NO: 71). The invention further relates to amethod for the preparation of a multivalent and/or multispecificcompound, construct or polypeptide comprising or essentially consistingof SEQ ID NO's: 70-72 (preferably SEQ ID NO's: 70-71, more preferablySEQ ID NO: 70 or SEQ ID NO: 71), comprising the linking of a monovalentconstruct comprising or essentially consisting of one of SEQ ID NO's:60-69 (preferably SEQ ID NO's: 65-69, more preferably SEQ ID NO: 66) toan amino acid sequence comprising or essential consisting of SEQ ID NO:98 via one or more linkers.

Suitable spacers or linkers for use in multivalent and/or multispecificpolypeptides will be clear to the skilled person, and may generally beany linker or spacer used in the art to link amino acid sequences.Preferably, said linker or spacer is suitable for use in constructingproteins or polypeptides that are intended for pharmaceutical use.

Some particularly preferred spacers include the spacers and linkers thatare used in the art to link antibody fragments or antibody domains.These include the linkers mentioned in the general background art citedabove, as well as for example linkers that are used in the art toconstruct diabodies or ScFv fragments (in this respect, however, itsshould be noted that, whereas in diabodies and in ScFv fragments, thelinker sequence used should have a length, a degree of flexibility andother properties that allow the pertinent V_(H) and V_(L) domains tocome together to form the complete antigen-binding site, there is noparticular limitation on the length or the flexibility of the linkerused in the polypeptide of the invention, since each amino acid sequenceor Nanobody by itself forms a complete antigen-binding site).

For example, a linker may be a suitable amino acid sequence, and inparticular amino acid sequences of between 1 and 50, preferably between1 and 30, such as between 1 and 20 or between 1 and 10 amino acidresidues. Some preferred examples of such amino acid sequences includegly-ser linkers, for example of the type (gly_(x)ser_(y))_(z), such as(for example (gly₄ser)₃ or (gly₃ser₂)₃, as described in WO 99/42077,hinge-like regions such as the hinge regions of naturally occurringheavy chain antibodies or similar sequences (such as described in WO94/04678).

Some other particularly preferred linkers are poly-alanine (such asAAA), as well as the linkers mentioned in Table B-8, of which AAA, GS-7and GS-9 are particularly preferred.

Other suitable linkers generally comprise organic compounds or polymers,in particular those suitable for use in proteins for pharmaceutical use.For instance, polyethyleneglycol) moieties have been used to linkantibody domains, see for example WO 04/081026.

It is encompassed within the scope of the invention that the length, thedegree of flexibility and/or other properties of the linker(s) used(although not critical, as it usually is for linkers used in ScFvfragments) may have some influence on the properties of the finalpolypeptide of the invention, including but not limited to the affinity,specificity or avidity for the IL-6 receptor, or for one or more of theother antigens. Based on the disclosure herein, the skilled person willbe able to determine the optimal linker(s) for use in a specificpolypeptide of the invention, optionally after some limited routineexperiments.

It is also within the scope of the invention that the linker(s) usedconfer one or more other favourable properties or functionality to thepolypeptides of the invention, and/or provide one or more sites for theformation of derivatives and/or for the attachment of functional groups(e.g. as described herein for the derivatives of the amino acidsequences, Nanobodies, compounds and polypeptides of the invention). Forexample, linkers containing one or more charged amino acid residues canprovide improved hydrophilic properties, whereas linkers that form orcontain small epitopes or tags can be used for the purposes ofdetection, identification and/or purification. Again, based on thedisclosure herein, the skilled person will be able to determine theoptimal linkers for use in a specific polypeptide of the invention,optionally after some limited routine experiments.

Finally, when two or more linkers are used in the polypeptides of theinvention, these linkers may be the same or different. Again, based onthe disclosure herein, the skilled person will be able to determine theoptimal linkers for use in a specific polypeptide of the invention,optionally after some limited routine experiments.

Usually, for easy of expression and production, a polypeptide of theinvention will be a linear polypeptide. However, the invention in itsbroadest sense is not limited thereto. For example, when a polypeptideof the invention comprises three of more amino acid sequences orNanobodies, it is possible to link them by use of a linker with three ormore “arms”, which each “arm” being linked to an amino acid sequence orNanobody, so as to provide a “star-shaped” construct. It is alsopossible, although usually less preferred, to use circular constructs.

The invention in its broadest sense also comprises derivatives of theamino acid sequences, Nanobodies, compounds or polypeptides of theinvention, Such derivatives can generally be obtained by modification,and in particular by chemical and/or biological (e.g. enzymatical)modification, of the amino acid sequences, Nanobodies, compounds orpolypeptides of the invention and/or of one or more of the amino acidresidues that form the amino acid sequences, Nanobodies, compounds orpolypeptides of the invention.

Examples of such modifications, as well as examples of amino acidresidues within the amino acid sequence, Nanobody sequence, compound orpolypeptide sequences that can be modified in such a manner (i.e. eitheron the protein backbone but preferably on a side chain), methods andtechniques that can be used to introduce such modifications and thepotential uses and advantages of such modifications will be clear to theskilled person.

For example, such a modification may involve the introduction (e.g. bycovalent linking or in an other suitable manner) of one or morefunctional groups, residues or moieties into or onto the amino acidsequence, Nanobody, compound or polypeptide of the invention, and inparticular of one or more functional groups, residues or moieties thatconfer one or more desired properties or functionalities to the aminoacid sequence, Nanobody, compound or polypeptide of the invention.Example of such functional groups will be clear to the skilled person.

For example, such modification may comprise the introduction (e.g. bycovalent binding or in any other suitable manner) of one or morefunctional groups that that increase the half-life, the solubilityand/or the absorption of the amino acid sequence, Nanobody, compound orpolypeptide of the invention, that reduce the immunogenicity and/or thetoxicity of the amino acid sequence, Nanobody, compound or polypeptideof the invention, that eliminate or attenuate any undesirable sideeffects of the amino acid sequence, Nanobody, compound or polypeptide ofthe invention, and/or that confer other advantageous properties toand/or reduce the undesired properties of the amino acid sequence,Nanobody, compound or polypeptide of the invention; or any combinationof two or more of the foregoing. Examples of such functional groups andof techniques for introducing them will be clear to the skilled person,and can generally comprise all functional groups and techniquesmentioned in the general background art cited hereinabove as well as thefunctional groups and techniques known per se for the modification ofpharmaceutical proteins, and in particular for the modification ofantibodies or antibody fragments (including ScFv's and single domainantibodies), for which reference is for example made to Remington'sPharmaceutical Sciences (1980, 16th ed., Mack Publishing Co., Easton,Pa.). Such functional groups may for example be linked directly (forexample covalently) to an amino acid sequence, Nanobody, compound orpolypeptide of the invention, or optionally via a suitable linker orspacer, as will again be clear to the skilled person.

One of the most widely used techniques for increasing the half-lifeand/or reducing the immunogenicity of pharmaceutical proteins comprisesattachment of a suitable pharmacologically acceptable polymer, such aspolyethyleneglycol) (PEG) or derivatives thereof (such asmethoxypoly(ethyleneglycol) or mPEG). Generally, any suitable form ofpegylation can be used, such as the pegylation used in the art forantibodies and antibody fragments (including but not limited to (single)domain antibodies and ScFv's); reference is made to for example Chapman(2002, Nat. Biotechnol., 54: 531-545); by Veronese and Harris (2003,Adv. Drug Deliv. Rev. 54: 453-456), by Harris and Chess (2003, Nat. Rev.Drug. Discov., 2: 214-21) and in WO 04/060955. Various reagents forpegylation of proteins are also commercially available, for example fromNektar Therapeutics, USA.

Preferably, site-directed pegylation is used, in particular via acysteine-residue (see for example Yang et al. (2003, ProteinEngineering, 16 (10): 761-770). For example, for this purpose, PEG maybe attached to a cysteine residue that naturally occurs in an amino acidsequence, Nanobody, compound or polypeptide of the invention, an aminoacid sequence, Nanobody, compound or polypeptide of the invention may bemodified so as to suitably introduce one or more cysteine residues forattachment of PEG, or an amino acid sequence comprising one or morecysteine residues for attachment of PEG may be fused to the N- and/orC-terminus of an amino acid sequence, Nanobody, compound or polypeptideof the invention, all using techniques of protein engineering known perse to the skilled person.

Preferably, for the amino acid sequences, Nanobodies, compounds orpolypeptides of the invention of the invention, a PEG is used with amolecular weight of more than 5000, such as more than 10,000 and lessthan 200,000, such as less than 100,000; for example in the range of20,000-80,000.

Another, usually less preferred modification comprises N-linked orO-linked glycosylation, usually as part of co-translational and/orpost-translational modification, depending on the host cell used forexpressing the amino acid sequence, Nanobody, compound or polypeptide ofthe invention.

Yet another modification may comprise the introduction of one or moredetectable labels or other signal-generating groups or moieties,depending on the intended use of the labelled amino acid sequence,Nanobody, compound or polypeptide of the invention. Suitable labels andtechniques for attaching, using and detecting them will be clear to theskilled person, and for example include, but are not limited to,fluorescent labels (such as fluorescein, isothiocyanate, rhodamine,phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, andfluorescamine and fluorescent metals such as ¹⁵²Eu or others metals fromthe lanthanide series), phosphorescent labels, chemiluminescent labelsor bioluminescent labels (such as luminal, isoluminol, theromaticacridinium ester, imidazole, acridinium salts, oxalate ester, dioxetaneor GIP and its analogs), radio-isotopes (such as ³H, ¹²⁵I, ³²P, ³⁵S,¹⁴C, ⁵¹Cr, ³⁶Cl, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, and ⁷⁵Se), metals, metals chelates ormetallic cations (for example metallic cations such as ^(99m)Tc, ¹²³I,¹¹¹In, ¹³¹I, ⁹⁷Ru, ⁶⁷Cu, ⁶⁷Ga, and ⁶⁸Ga or other metals or metalliccations that are particularly suited for use in in vivo, in vitro or insitu diagnosis and imaging, such as (¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Cr, and⁵⁶Fe), as well as chromophores and enzymes (such as malatedehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeastalcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triosephosphate isomerase, biotinavidin peroxidase, horseradish peroxidase,alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase,ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase,glucoamylase and acetylcholine esterase). Other suitable labels will beclear to the skilled person, and for example include moieties that canbe detected using NMR or ESR spectroscopy.

Such labelled amino acid sequences, Nanobodies, compounds orpolypeptides of the invention may for example be used for in vitro, invivo or in situ assays (including immunoassays known per se such asELISA, RIA, EIA and other “sandwich assays”, etc.) as well as in vivodiagnostic and imaging purposes, depending on the choice of the specificlabel.

As will be clear to the skilled person, another modification may involvethe introduction of a chelating group, for example to chelate one of themetals or metallic cations referred to above. Suitable chelating groupsfor example include, without limitation, diethyl-enetriaminepentaaceticacid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

Yet another modification may comprise the introduction of a functionalgroup that is one part of a specific binding pair, such as thebiotin-(strept)avidin binding pair. Such a functional group may be usedto link the amino acid sequence, Nanobody, compound or polypeptide ofthe invention to another protein, polypeptide or chemical compound thatis bound to the other half of the binding pair, i.e. through formationof the binding pair. For example, an amino acid sequence, Nanobody,compound or polypeptide of the invention may be conjugated to biotin,and linked to another protein, polypeptide, compound or carrierconjugated to avidin or streptavidin. For example, such a conjugatedamino acid sequence, Nanobody, compound or polypeptide of the inventionmay be used as a reporter, for example in a diagnostic system where adetectable signal-producing agent is conjugated to avidin orstreptavidin. Such binding pairs may for example also be used to bindthe amino acid sequence, Nanobody, compound or polypeptide of theinvention to a carrier, including carriers suitable for pharmaceuticalpurposes. One non-limiting example are the liposomal formulationsdescribed by Cao and Suresh (2000, Journal of Drug Targeting, 8 (4):257). Such binding pairs may also be used to link a therapeuticallyactive agent to the amino acid sequence, Nanobody, compound orpolypeptide of the invention.

Other potential chemical and enzymatical modifications will be clear tothe skilled person. Such modifications may also be introduced forresearch purposes (e.g. to study function-activity relationships).Reference is for example made to Lundblad and Bradshaw (1997,Biotechnol. Appl. Biochem., 26: 143-151).

Preferably, the derivatives are such that they bind to the specificeptiope on the IL-6 receptor, with an affinity (suitably measured and/orexpressed as a K_(D)-value (actual or apparent), a K_(A)-value (actualor apparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively asan IC₅₀ value, as further described herein) that is as defined herein.

In particular, such derivatives of the invention are preferably suchthat they:

-   -   bind to hIL-6R with a dissociation constant (K_(D)) of 1 nM to 1        pM moles/litre or less, preferably 500 pM to 1 pM moles/litre or        less, more preferably 100 pM to 1 pM moles/litre or less, or        even more preferably about 50 pM to 1 pM or less;        and/or such that they:    -   bind to cyno IL-6R with a dissociation constant (K_(D)) of 1 nM        to 1 pM moles/litre or less, preferably 500 pM to 1 pM        moles/litre or less, more preferably 100 pM to 1 pM moles/litre        or less, or even more preferably about 50 pM to 1 pM or less;        and/or such that they:    -   bind to hIL-6R with a k_(on)-rate of between 10⁴ M⁻¹s⁻¹ to about        10⁷ M⁻¹s⁻¹, preferably between 10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more        preferably about 10⁶ M⁻¹s⁻¹ or more;        and/or such that they:    -   bind to cyno IL-6R with a k_(on)-rate of between 10⁴ M⁻¹s⁻¹ to        about 10⁷ M⁻¹s⁻¹, preferably between 10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹,        more preferably about 10⁵ M⁻¹s⁻¹ or more;        and/or such that they:    -   bind to hIL-6R with a k_(off) rate between 10⁻³ (t_(1/2)=0.69 s)        and 10⁻⁶ s⁻¹ (providing a near irreversible complex with a        t_(1/2) of multiple days), preferably between 10⁻⁴ s⁻¹ and 10⁻⁶        s⁻¹, more preferably between 10⁻⁵ s⁻¹ and 10⁻⁶ s⁻¹, such as        about 10⁻⁵ s⁻¹ or lower;        and/or such that they:    -   bind to cyno IL-6R with a k_(off) rate between 10⁻³ s⁻¹        (t_(1/2)=0.69 s) and 10⁻⁶ s⁻¹ (providing a near irreversible        complex with a t_(1/2) of multiple days), preferably between        10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹, more preferably between 10⁻⁵ s⁻¹ and 10⁻⁶        s⁻¹, such as about 10⁻⁵ s⁻¹ or lower.

As mentioned above, the invention also relates to proteins orpolypeptides that essentially consist of or comprise at least one aminoacid sequence, Nanobody, compound or polypeptide of the invention. By“essentially consist of” is meant that the amino acid sequence of theprotein or polypeptide of the invention either is exactly the same asthe amino acid sequence, Nanobody, compound or polypeptide of theinvention or corresponds to the amino acid sequence, Nanobody, compoundor polypeptide of the invention which has a limited number of amino acidresidues, such as 1-20 amino acid residues, for example 1-10 amino acidresidues and preferably 1-6 amino acid residues, such as 1, 2, 3, 4, 5or 6 amino acid residues, added at the amino terminal end, at thecarboxy terminal end, or at both the amino terminal end and the carboxyterminal end of the amino acid sequence, Nanobody, compound orpolypeptide.

Said amino acid residues may or may not change, alter or otherwiseinfluence the (biological) properties of the amino acid sequence,Nanobody, compound or polypeptide of the invention and may or may notadd further functionality to the amino acid sequence, Nanobody, compoundor polypeptide. For example, such amino acid residues:

-   a) can comprise an N-terminal Met residue, for example as result of    expression in a heterologous host cell or host organism.-   b) may form a signal sequence or leader sequence that directs    secretion of the amino acid sequence, Nanobody, compound or    polypeptide from a host cell upon synthesis. Suitable secretory    leader peptides will be clear to the skilled person, and may be as    further described herein. Usually, such a leader sequence will be    linked to the N-terminus of the amino acid sequence, Nanobody,    compound or polypeptide, although the invention in its broadest    sense is not limited thereto;-   c) may form a sequence or signal that allows the amino acid    sequence, Nanobody, compound or polypeptide to be directed towards    and/or to penetrate or enter into specific organs, tissues, cells,    or parts or compartments of cells, and/or that allows the amino acid    sequence, Nanobody, compound or polypeptide to penetrate or cross a    biological barrier such as a cell membrane, a cell layer such as a    layer of epithelial cells, a tumor including solid tumors, or the    blood-brain-barrier. Examples of such amino acid sequences will be    clear to the skilled person. Some non-limiting examples are the    small peptide vectors (“Pep-trans vectors”) described in WO    03/026700 and in Temsamani et al., Expert Opin. Biol. Ther., 1, 773    (2001); Temsamani and Vidal, Drug Discov. Today, 9, 1012 (004) and    Rousselle, J. Pharmacol. Exp. Ther., 296, 124-131 (2001), and the    membrane translocator sequence described by Zhao et al., Apoptosis,    8, 631-637 (2003). C-terminal and N-terminal amino acid sequences    for intracellular targeting of antibody fragments are for example    described by Cardinale et al., Methods, 34, 171 (2004). Other    suitable techniques for intracellular targeting involve the    expression and/or use of so-called “intrabodies” comprising a amino    acid sequence, Nanobody, compound or polypeptide of the invention,    as mentioned below;-   d) may form a “tag”, for example an amino acid sequence or residue    that allows or facilitates the purification of the amino acid    sequence, Nanobody, compound or polypeptide, for example using    affinity techniques directed against said sequence or residue.    Thereafter, said sequence or residue may be removed (e.g. by    chemical or enzymatical cleavage) to provide the amino acid    sequence, Nanobody, compound or polypeptide (for this purpose, the    tag may optionally be linked to the amino acid sequence, Nanobody,    compound or polypeptide sequence via a cleavable linker sequence or    contain a cleavable motif). Some preferred, but non-limiting    examples of such residues are multiple histidine residues,    glutatione residues and a myc-tag such as AAAEQKLISEEDLNGAA (SEQ ID    NO: 100);-   e) may be one or more amino acid residues that have been    functionalized and/or that can serve as a site for attachment of    functional groups. Suitable amino acid residues and functional    groups will be clear to the skilled person and include, but are not    limited to, the amino acid residues and functional groups mentioned    herein for the derivatives of the amino acid sequences, Nanobodies,    compounds or polypeptides of the invention.

The amino acid sequences, Nanobodies, polypeptides and nucleic acids ofthe invention can be prepared in a manner known per se, as will be clearto the skilled person from the further description herein. For example,the amino acid sequences, Nanobodies and polypeptides of the inventioncan be prepared in any manner known per se for the preparation ofantibodies and in particular for the preparation of antibody fragments(including but not limited to (single) domain antibodies and ScFvfragments). Some preferred, but non-limiting methods for preparing theamino acid sequences, Nanobodies, polypeptides and nucleic acids includethe methods and techniques described herein.

As will be clear to the skilled person, one particularly useful methodfor preparing an amino acid sequence, Nanobody and/or a polypeptide ofthe invention generally comprises the steps of:

-   -   the expression, in a suitable host cell or host organism (also        referred to herein as a “host of the invention”) or in another        suitable expression system of a nucleic acid that encodes said        amino acid sequence, Nanobody or polypeptide of the invention        (also referred to herein as a “nucleic acid of the invention”),        optionally followed by:    -   isolating and/or purifying the amino acid sequence, Nanobody or        polypeptide of the invention thus obtained.

In particular, such a method may comprise the steps of:

-   -   cultivating and/or maintaining a host of the invention under        conditions that are such that said host of the invention        expresses and/or produces at least one amino acid sequence,        Nanobody and/or polypeptide of the invention; optionally        followed by:    -   isolating and/or purifying the amino acid sequence, Nanobody or        polypeptide of the invention thus obtained.

Accordingly, the present invention also relates to a nucleic acid ornucleotide sequence that encodes an amino acid sequence, a Nanobody, apolypeptide or a monovalent construct of the invention (also referred toas “nucleic acid of the invention” or “nucleotide sequence of theinvention”). A nucleic acid of the invention can be in the form ofsingle or double stranded DNA or RNA, and is preferably in the form ofdouble stranded DNA. For example, the nucleotide sequences of theinvention may be genomic DNA, cDNA or synthetic DNA (such as DNA with acodon usage that has been specifically adapted for expression in theintended host cell or host organism).

According to one embodiment of the invention, the nucleic acid of theinvention is in essentially isolated from, as defined herein. Thenucleic acid of the invention may also be in the form of, be present inand/or be part of a vector, such as for example a plasmid, cosmid orYAC, which again may be in essentially isolated form.

The nucleic acids of the invention can be prepared or obtained in amanner known per se, based on the information on the amino acidsequences, Nanobodies and/or polypeptides of the invention given herein,and/or can be isolated from a suitable natural source. Also, as will beclear to the skilled person, to prepare a nucleic acid of the invention,also several nucleotide sequences, such as at least one nucleotidesequence encoding an amino acid sequence or Nanobody and for examplenucleic acids encoding one or more linkers can be linked together in asuitable manner.

Techniques for generating the nucleic acids of the invention will beclear to the skilled person and may for instance include, but are notlimited to, automated DNA synthesis; site-directed mutagenesis;combining two or more naturally occurring and/or synthetic sequences (ortwo or more parts thereof), introduction of mutations that lead to theexpression of a truncated expression product; introduction of one ormore restriction sites (e.g. to create cassettes and/or regions that mayeasily be digested and/or ligated using suitable restriction enzymes),and/or the introduction of mutations by means of a PCR reaction usingone or more “mismatched” primers. These and other techniques will beclear to the skilled person, and reference is again made to the standardhandbooks, such as Sambrook et al. and Ausubel et al., mentioned above,as well as the Examples below.

The nucleic acid of the invention may also be in the form of, be presentin and/or be part of a genetic construct, as will be clear to the personskilled in the art. Such genetic constructs generally comprise at leastone nucleic acid of the invention that is optionally linked to one ormore elements of genetic constructs known per se, such as for exampleone or more suitable regulatory elements (such as a suitablepromoter(s), enhancer(s), terminator(s), etc.) and the further elementsof genetic constructs referred to herein. Such genetic constructscomprising at least one nucleic acid of the invention will also bereferred to herein as “genetic constructs of the invention”.

The genetic constructs of the invention may be DNA or RNA, and arepreferably double-stranded DNA. The genetic constructs of the inventionmay also be in a form suitable for transformation of the intended hostcell or host organism, in a form suitable for integration into thegenomic DNA of the intended host cell or in a form suitable forindependent replication, maintenance and/or inheritance in the intendedhost organism. For instance, the genetic constructs of the invention maybe in the form of a vector, such as for example a plasmid, cosmid, YAC,a viral vector or transposon. In particular, the vector may be anexpression vector, i.e. a vector that can provide for expression invitro and/or in vivo (e.g. in a suitable host cell, host organism and/orexpression system).

In a preferred but non-limiting embodiment, a genetic construct of theinvention comprises

a) at least one nucleic acid of the invention; operably connected tob) one or more regulatory elements, such as a promoter and optionally asuitable terminator;and optionally alsoc) one or more further elements of genetic constructs known per se;

in which the terms “regulatory element”, “promoter”, “terminator” and“operably connected” have their usual meaning in the art (as furtherdescribed herein); and in which said “further elements” present in thegenetic constructs may for example be 3′- or 5′-UTR sequences, leadersequences, selection markers, expression markers/reporter genes, and/orelements that may facilitate or increase (the efficiency of)transformation or integration. These and other suitable elements forsuch genetic constructs will be clear to the skilled person, and may forinstance depend upon the type of construct used, the intended host cellor host organism; the manner in which the nucleotide sequences of theinvention of interest are to be expressed (e.g. via constitutive,transient or inducible expression); and/or the transformation techniqueto be used. For example, regulatory sequences, promoters and terminatorsknown per se for the expression and production of antibodies andantibody fragments (including but not limited to (single) domainantibodies and ScFv fragments) may be used in an essentially analogousmanner.

Preferably, in the genetic constructs of the invention, said at leastone nucleic acid of the invention and said regulatory elements, andoptionally said one or more further elements, are “operably linked” toeach other, by which is generally meant that they are in a functionalrelationship with each other. For instance, a promoter is considered“operably linked” to a coding sequence if said promoter is able toinitiate or otherwise control/regulate the transcription and/or theexpression of a coding sequence (in which said coding sequence should beunderstood as being “under the control of” said promotor). Generally,when two nucleotide sequences are operably linked, they will be in thesame orientation and usually also in the same reading frame. They willusually also be essentially contiguous, although this may also not berequired.

Preferably, the regulatory and further elements of the geneticconstructs of the invention are such that they are capable of providingtheir intended biological function in the intended host cell or hostorganism.

For instance, a promoter, enhancer or terminator should be “operable” inthe intended host cell or host organism, by which is meant that (forexample) said promoter should be capable of initiating or otherwisecontrolling/regulating the transcription and/or the expression of anucleotide sequence—e.g. a coding sequence—to which it is operablylinked (as defined herein).

Some particularly preferred promoters include, but are not limited to,promoters known per se for the expression in the host cells mentionedherein; and in particular promoters for the expression in the bacterialcells, such as those mentioned herein and/or those used in the Examples.

A selection marker should be such that it allows—i.e. under appropriateselection conditions—host cells and/or host organisms that have been(successfully) transformed with the nucleotide sequence of the inventionto be distinguished from host cells/organisms that have not been(successfully) transformed. Some preferred, but non-limiting examples ofsuch markers are genes that provide resistance against antibiotics (suchas kanamycin or ampicillin), genes that provide for temperatureresistance, or genes that allow the host cell or host organism to bemaintained in the absence of certain factors, compounds and/or (food)components in the medium that are essential for survival of thenon-transformed cells or organisms.

A leader sequence should be such that—in the intended host cell or hostorganism—it allows for the desired post-translational modificationsand/or such that it directs the transcribed mRNA to a desired part ororganelle of a cell. A leader sequence may also allow for secretion ofthe expression product from said cell. As such, the leader sequence maybe any pro-, pre-, or prepro-sequence operable in the host cell or hostorganism. Leader sequences may not be required for expression in abacterial cell. For example, leader sequences known per se for theexpression and production of antibodies and antibody fragments(including but not limited to single domain antibodies and ScFvfragments) may be used in an essentially analogous manner.

An expression marker or reporter gene should be such that—in the hostcell or host organism—it allows for detection of the expression of (agene or nucleotide sequence present on) the genetic construct. Anexpression marker may optionally also allow for the localisation of theexpressed product, e.g. in a specific part or organelle of a cell and/orin (a) specific cell(s), tissue(s), organ(s) or part(s) of amulticellular organism. Such reporter genes may also be expressed as aprotein fusion with the amino acid sequence, Nanobody or polypeptide ofthe invention. Some preferred, but non-limiting examples includefluorescent proteins such as GFP.

Some preferred, but non-limiting examples of suitable promoters,terminator and further elements include those that can be used for theexpression in the host cells mentioned herein; and in particular thosethat are suitable for expression in bacterial cells, such as thosementioned herein and/or those used in the Examples below. For some(further) non-limiting examples of the promoters, selection markers,leader sequences, expression markers and further elements that may bepresent/used in the genetic constructs of the invention—such asterminators, transcriptional and/or translational enhancers and/orintegration factors—reference is made to the general handbooks such asSambrook et al. and Ausubel et al. mentioned above, as well as to theexamples that are given in WO 95/07463, WO 96/23810, WO 95/07463, WO95/21191, WO 97/11094, WO 97/42320, WO 98/06737, WO 98/21355, U.S. Pat.No. 7,207,410, U.S. Pat. No. 5,593,492 and EP 1085089. Other exampleswill be clear to the skilled person. Reference is also made to thegeneral background art cited above and the further references citedherein.

The genetic constructs of the invention may generally be provided bysuitably linking the nucleotide sequence(s) of the invention to the oneor more further elements described above, for example using thetechniques described in the general handbooks such as Sambrook et al.and Ausubel et al., mentioned above.

Often, the genetic constructs of the invention will be obtained byinserting a nucleotide sequence of the invention in a suitable(expression) vector known per se. Some preferred, but non-limitingexamples of suitable expression vectors are those used in the Examplesbelow, as well as those mentioned herein.

The nucleic acids of the invention and/or the genetic constructs of theinvention may be used to transform a host cell or host organism, i.e.for expression and/or production of the amino acid sequence, Nanobody orpolypeptide of the invention. Suitable hosts or host cells will be clearto the skilled person, and may for example be any suitable fungal,prokaryotic or eukaryotic cell or cell line or any suitable fungal,prokaryotic or eukaryotic organism, for example:

-   -   a bacterial strain, including but not limited to gram-negative        strains such as strains of Escherichia coli; of Proteus, for        example of Proteus mirabilis; of Pseudomonas, for example of        Pseudomonas fluorescens; and gram-positive strains such as        strains of Bacillus, for example of Bacillus subtilis or of        Bacillus brevis; of Streptomyces, for example of Streptomyces        llyidans; of Staphylococcus, for example of Staphylococcus        carnasus; and of Lactococcus, for example of Lactococcus lactis;    -   a fungal cell, including but not limited to cells from species        of Trichoderma, for example from Trichoderma reesei; of        Neurospora, for example from Neurospora crassa; of Sordaria, for        example from Sordaria macrospora; of Aspergillus, for example        from Aspergillus niger or from Aspergillus sojae; or from other        filamentous fungi;    -   a yeast cell, including but not limited to cells from species of        Saccharomyces, for example of Saccharomyces cerevisiae; of        Schizosocchoromyces, for example of Schizosaccharomyces pombe;        of Pichia, for example of Pichia pastoris or of Pichia        methanolica; of Hansenula, for example of Hansenula polymorpha;        of Kluyveromyces, for example of Kluyveromyces lactis; of        Arxula, for example of Arxula adeninivorans; of Yarrowia, for        example of Yarrowia lipolytica;    -   an amphibian cell or cell line, such as Xenopus oocytes;    -   an insect-derived cell or cell line, such as cells/cell lines        derived from lepidoptera, including but not limited to        Spodoptera SF9 and Sf21 cells or cells/cell lines derived from        Drosophila, such as Schneider and Kc cells;    -   a plant or plant cell, for example in tobacco plants; and/or    -   a mammalian cell or cell line, for example a cell or cell line        derived from a human, a cell or a cell line from mammals        including but not limited to CHO-cells, MK-cells (for example        MK-21 cells) and human cells or cell lines such as HeLa, COS        (for example COS-7) and PER.C6 cells;        as well as all other hosts or host cells known per se for the        expression and production of antibodies and antibody fragments        (including but not limited to (single) domain antibodies and        ScFv fragments), which will be clear to the skilled person.        Reference is also made to the general background art cited        hereinabove, as well as to for example WO 94/29457; WO 96/34103;        WO 99/42077; Frenken et al. (1998, Res. Immunol. 149(6):        589-99), Riechmann and Muyldermans (1999, J. Immunol. Methods,        231 (1-2): 25-38), van der Linden (2000, J. Biotechnol. 80(3):        261-70), Joosten et al. (2003, Microb. Cell Fact. 2(1): 1),        Joosten et al. (2005, Appl. Microbiol, Biotechnol. 66(4):        384-92); and the further references cited herein.

The amino acid sequences, Nanobodies and polypeptides of the inventioncan also be introduced and expressed in one or more cells, tissues ororgans of a multicellular organism, for example for prophylactic and/ortherapeutic purposes (e.g. as a gene therapy). For this purpose, thenucleotide sequences of the invention may be introduced into the cellsor tissues in any suitable way, for example as such (e.g. usingliposomes) or after they have been inserted into a suitable gene therapyvector (for example derived from retroviruses such as adenovirus, orparvoviruses such as adeno-associated virus). As will also be clear tothe skilled person, such gene therapy may be performed in vivo and/or insitu in the body of a patient by administering a nucleic acid of theinvention or a suitable gene therapy vector encoding the same to thepatient or to specific cells or a specific tissue or organ of thepatient; or suitable cells (often taken from the body of the patient tobe treated, such as explanted lymphocytes, bone marrow aspirates ortissue biopsies) may be treated in vitro with a nucleotide sequence ofthe invention and then be suitably (re-)introduced into the body of thepatient. All this can be performed using gene therapy vectors,techniques and delivery systems which are well known to the skilledperson, and for example described in Culver K. W, (1994, “Gene Therapy”,p. xii, Mary Ann Liebert, Inc., Publishers, New York, N.Y.), Giordano(1996, Nature F Medicine 2: 534-539), Schaper (1996, Circ. Res. 79:911-919), Anderson (1992, Science 256: 808-813), Verma (1994, Nature389: 239); lsner (1996, Lancet 348: 370-374), Muhlhauser (1995, Circ.Res. 77: 1077-1086); Onodera (1998, Blood 91: 30-36); Verma (1998, GeneTher. 5: 692-699); Nabel (1997, Ann. N.Y. Acad. Sci., 811: 289-292),Verzeletti (1998, Hum. Gene Ther. 9: 2243-51); Wang 1996, NatureMedicine 2: 714-716), WO 94/29469, WO 97/00957, U.S. Pat. No. 5,580,859,or Schaper (1996, Current Opinion in Biotechnology 7: 635-640). Forexample, in situ expression of ScFv fragments (Afanasieva et al. (2003,Gene Ther., 10: 1850-1859) and of diabodies (Blanco et al., 2003, J.Immunol., 171: 1070-1077) has been described in the art.

For expression of the amino acid sequences, Nanobodies or polypeptidesin a cell, they may also be expressed as so-called “intrabodies”, as forexample described in WO 94/02610, WO 95/22618, U.S. Pat. No. 7,004,940,WO 03/014960, in Cattaneo A. and Biocca S. (1997, intracellularAntibodies: Development and Applications. Landes and Springer-Verlag)and in Kontermann (2004, Methods 34: 163-170).

The amino acid sequences, Nanobodies and polypeptides of the inventioncan for example also be produced in the milk of transgenic mammals, forexample in the milk of rabbits, cows, goats or sheep (see for exampleU.S. Pat. No. 6,741,957, U.S. Pat. No. 6,304,489 and U.S. Pat. No.6,849,992 for general techniques for introducing transgenes intomammals), in plants or parts of plants including but not limited totheir leaves, flowers, fruits, seed, roots or turbers (for example intobacco, maize, soybean or alfalfa) or in for example pupae of thesilkworm Bombix mori.

Furthermore, the amino acid sequences, Nanobodies and polypeptides ofthe invention can also be expressed and/or produced in cell-freeexpression systems, and suitable examples of such systems will be clearto the skilled person. Some preferred, but non-limiting examples includeexpression in the wheat germ system; in rabbit reticulocyte lysates; orin the E. coli Zubay system.

As mentioned above, one of the advantages of the use of Nanobodies isthat the polypeptides based thereon can be prepared through expressionin a suitable bacterial system, and suitable bacterial expressionsystems, vectors, host cells, regulatory elements, etc., will be clearto the skilled person, for example from the references cited above. Itshould however be noted that the invention in its broadest sense is notlimited to expression in bacterial systems.

Preferably, in the invention, an (in vivo or in vitro) expressionsystem, such as a bacterial expression system, is used that provides thepolypeptides of the invention in a form that is suitable forpharmaceutical use, and such expression systems will again be clear tothe skilled person. As also will be clear to the skilled person,polypeptides of the invention suitable for pharmaceutical use can beprepared using techniques for peptide synthesis.

For production on industrial scale, preferred heterologous hosts for the(industrial) production of Nanobodies or Nanobody-containing proteintherapeutics include strains of E. coli, Pichia pastoris, S. cerevisiaethat are suitable for large scale expression/production/fermentation,and in particular for large scale pharmaceuticalexpression/production/fermentation. Suitable examples of such strainswill be clear to the skilled person. Such strains andproduction/expression systems are also made available by companies suchas Biovitrum (Uppsala, Sweden).

Alternatively, mammalian cell lines, in particular Chinese hamster ovary(CHO) cells, can be used for large scaleexpression/production/fermentation, and in particular for large scalepharmaceutical expression/production/fermentation. Again, suchexpression/production systems are also made available by some of thecompanies mentioned above.

The choice of the specific expression system would depend in part on therequirement for certain post-translational modifications, morespecifically glycosylation. The production of a Nanobody-containingrecombinant protein for which glycosylation is desired or required wouldnecessitate the use of mammalian expression hosts that have the abilityto glycosylate the expressed protein. In this respect, it will be clearto the skilled person that the glycosylation pattern obtained (i.e. thekind, number and position of residues attached) will depend on the cellor cell line that is used for the expression. Preferably, either a humancell or cell line is used (i.e. leading to a protein that essentiallyhas a human glycosylation pattern) or another mammalian cell line isused that can provide a glycosylation pattern that is essentially and/orfunctionally the same as human glycosylation or at least mimics humanglycosylation. Generally, prokaryotic hosts such as E. coli do not havethe ability to glycosylate proteins, and the use of lower eukaryotessuch as yeast usually leads to a glycosylation pattern that differs fromhuman glycosylation. Nevertheless, it should be understood that all theforegoing host cells and expression systems can be used in theinvention, depending on the desired amino acid sequence, Nanobody orpolypeptide to be obtained.

Thus, according to one non-limiting embodiment of the invention, theamino acid sequence, Nanobody or polypeptide of the invention isglycosylated. According to another non-limiting embodiment of theinvention, the amino acid sequence, Nanobody or polypeptide of theinvention is non-glycosylated.

According to one preferred, but non-limiting embodiment of theinvention, the amino acid sequence, Nanobody or polypeptide of theinvention is produced in a bacterial cell, in particular a bacterialcell suitable for large scale pharmaceutical production, such as cellsof the strains mentioned above.

According to another preferred, but non-limiting embodiment of theinvention, the amino acid sequence, Nanobody or polypeptide of theinvention is produced in a yeast cell, in particular a yeast cellsuitable for large scale pharmaceutical production, such as cells of thespecies mentioned above.

According to yet another preferred, but non-limiting embodiment of theinvention, the amino acid sequence, Nanobody or polypeptide of theinvention is produced in a mammalian cell, in particular in a human cellor in a cell of a human cell line, and more in particular in a humancell or in a cell of a human cell line that is suitable for large scalepharmaceutical production, such as the cell lines mentioned hereinabove.

When expression in a host cell is used to produce the amino acidsequences, Nanobodies and the polypeptides of the invention, the aminoacid sequences, Nanobodies and polypeptides of the invention can beproduced either intracellularly (e.g. in the cytosol, in the periplasmaor in inclusion bodies) and then isolated from the host cells andoptionally further purified; or can be produced extracellularly (e.g. inthe medium in which the host cells are cultured) and then isolated fromthe culture medium and optionally further purified. When eukaryotic hostcells are used, extracellular production is usually preferred since thisconsiderably facilitates the further isolation and downstream processingof the amino acid sequences, Nanobodies, polypeptides and proteinsobtained. Bacterial cells such as the strains of E. coli mentioned abovenormally do not secrete proteins extracellularly, except for a fewclasses of proteins such as toxins and hemolysin, and secretoryproduction in E. coli refers to the translocation of proteins across theinner membrane to the periplasmic space. Periplasmic production providesseveral advantages over cytosolic production. For example, theN-terminal amino acid sequence of the secreted product can be identicalto the natural gene product after cleavage of the secretion signalsequence by a specific signal peptidase. Also, there appears to be muchless protease activity in the periplasm than in the cytoplasm. Inaddition, protein purification is simpler due to fewer contaminatingproteins in the periplasm. Another advantage is that correct disulfidebonds may form because the periplasm provides a more oxidativeenvironment than the cytoplasm. Proteins overexpressed in E. coli areoften found in insoluble aggregates, so-called inclusion bodies. Theseinclusion bodies may be located in the cytosol or in the periplasm; therecovery of biologically active proteins from these inclusion bodiesrequires a denaturation/refolding process. Many recombinant proteins,including therapeutic proteins, are recovered from inclusion bodies.Alternatively, as will be clear to the skilled person, recombinantstrains of bacteria that have been genetically modified so as to secretea desired protein, and in particular a amino acid sequence, Nanobody ora polypeptide of the invention, can be used.

Thus, according to one non-limiting embodiment of the invention, theamino acid sequence, Nanobody or polypeptide of the invention is anamino acid sequence, Nanobody or polypeptide that has been producedintracellularly and that has been isolated from the host cell, and inparticular from a bacterial cell or from an inclusion body in abacterial cell. According to another non-limiting embodiment of theinvention, the amino acid sequence, Nanobody or polypeptide of theinvention is an amino acid sequence, Nanobody or polypeptide that hasbeen produced extracellularly, and that has been isolated from themedium in which the host cell is cultivated.

Some preferred, but non-limiting promoters for use with these host cellsinclude,

-   -   for expression in E. coli: lac promoter (and derivatives thereof        such as the lacUV5 promoter); arabinose promoter; left- (PL) and        rightward (PR) promoter of phage lambda; promoter of the trp        operon; hybrid lac/trp promoters (tac and trc); T7-promoter        (more specifically that of T7-phage gene 10) and other T-phage        promoters; promoter of the Tn10 tetracycline resistance gene;        engineered variants of the above promoters that include one or        more copies of an extraneous regulatory operator sequence;    -   for expression in S. cerevisiae: constitutive: ADH1 (alcohol        dehydrogenase 1), ENO (enolase), CYC1 (cytochrome c iso-1),        GAPDH (glyceraldehydes-3-phosphate dehydrogenase), PGK1        (phosphoglycerate kinase), PYK1 (pyruvate kinase); regulated:        GAL1,10,7 (galactose metabolic enzymes), ADH2 (alcohol        dehydrogenase 2), PHO5 (acid phosphatase), CUP1 (copper        metallothionein); heterologous: CaMV (cauliflower mosaic virus        35S promoter);    -   for expression in Pichia pastoris: the AOX1 promoter (alcohol        oxidase I);    -   for expression in mammalian cells: human cytomegalovirus (hCMV)        immediate early enhancer/promoter; human cytomegalovirus (hCMV)        immediate early promoter variant that contains two tetracycline        operator sequences such that the promoter can be regulated by        the Tet repressor; Herpes Simplex Virus thymidine kinase (TK)        promoter; Rous Sarcoma Virus long terminal repeat (RSV LTR)        enhancer/promoter; elongation factor 1α (hEF-1α) promoter from        human, chimpanzee, mouse or rat; the SV40 early promoter; HIV-1        long terminal repeat promoter; β-actin promoter;

Some preferred, but non-limiting vectors for use with these host cellsinclude:

-   -   vectors for expression in mammalian cells: pMAMneo (Clontech),        pcDNA3 (Invitrogen), pMC1neo (Stratagene), pSG5 (Stratagene),        EBO-pSV2-neo (ATCC 37593), pBPV-1 (8-2) (ATCC 37110),        pdBPV-MMTneo (342-12) (ATCC 37224), pRSVgpt (ATCC37199), pRSVneo        (ATCC37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460) and        1ZD35 (ATCC 37565), as well as viral-based expression systems,        such as those based on adenovirus;    -   vectors for expression in bacterial cells: pET vectors (Novagen)        and pQE vectors (Qiagen);    -   vectors for expression in yeast or other fungal cells: pYES2        (Invitrogen) and Pichia expression vectors (Invitrogen);    -   vectors for expression in insect cells: pBlueBacII (Invitrogen)        and other baculovirus vectors    -   vectors for expression in plants or plant cells: for example        vectors based on cauliflower mosaic virus or tobacco mosaic        virus, suitable strains of Agrobacterium, or Ti-plasmid based        vectors.

Some preferred, but non-limiting secretory sequences for use with thesehost cells include:

-   -   for use in bacterial cells such as E. coli: PelB, Bla, OmpA,        OmpC, OmpF, OmpT, StlI, PhoA, PhoE, MalE, Lpp, LamB, and the        like; TAT signal peptide, hemolysin C-terminal secretion signal;    -   for use in yeast: α-mating factor prepro-sequence, phosphatase        (pho1), invertase (Suc), etc.;    -   for use in mammalian cells: indigenous signal in case the target        protein is of eukaryotic origin; murine Ig κ-chain V-J2-C signal        peptide; etc.

Suitable techniques for transforming a host or host cell of theinvention will be clear to the skilled person and may depend on theintended host cell/host organism and the genetic construct to be used.Reference is again made to the handbooks and patent applicationsmentioned above.

After transformation, a step for detecting and selecting those hostcells or host organisms that have been successfully transformed with thenucleotide sequence/genetic construct of the invention may be performed.This may for instance be a selection step based on a selectable markerpresent in the genetic construct of the invention or a step involvingthe detection of the amino acid sequence of the invention, e.g. usingspecific antibodies.

The transformed host cell (which may be in the form or a stable cellline) or host organisms (which may be in the form of a stable mutantline or strain) form further aspects of the present invention.

Preferably, these host cells or host organisms are such that theyexpress, or are (at least) capable of expressing (e.g. under suitableconditions), an amino acid sequence, Nanobody or polypeptide of theinvention (and in case of a host organism: in at least one cell, part,tissue or organ thereof). The invention also includes furthergenerations, progeny and/or offspring of the host cell or host organismof the invention, that may for instance be obtained by cell division orby sexual or asexual reproduction.

To produce/obtain expression of the amino acid sequences of theinvention, the transformed host cell or transformed host organism maygenerally be kept, maintained and/or cultured under conditions such thatthe (desired) amino acid sequence, Nanobody or polypeptide of theinvention is expressed/produced. Suitable conditions will be clear tothe skilled person and will usually depend upon the host cell/hostorganism used, as well as on the regulatory elements that control theexpression of the (relevant) nucleotide sequence of the invention.Again, reference is made to the handbooks and patent applicationsmentioned above in the paragraphs on the genetic constructs of theinvention.

Generally, suitable conditions may include the use of a suitable medium,the presence of a suitable source of food and/or suitable nutrients, theuse of a suitable temperature, and optionally the presence of a suitableinducing factor or compound (e.g. when the nucleotide sequences of theinvention are under the control of an inducible promoter); all of whichmay be selected by the skilled person. Again, under such conditions, theamino acid sequences of the invention may be expressed in a constitutivemanner, in a transient manner, or only when suitably induced.

It will also be clear to the skilled person that the amino acidsequence, Nanobody or polypeptide of the invention may (first) begenerated in an immature form (as mentioned above), which may then besubjected to post-translational modification, depending on the hostcell/host organism used. Also, the amino acid sequence, Nanobody orpolypeptide of the invention may be glycosylated, again depending on thehost cell/host organism used.

The amino acid sequence, Nanobody or polypeptide of the invention maythen be isolated from the host cell/host organism and/or from the mediumin which said host cell or host organism was cultivated, using proteinisolation and/or purification techniques known per se, such as(preparative) chromatography and/or electrophoresis techniques,differential precipitation techniques, affinity techniques (e.g., usinga specific, cleavable amino acid sequence fused with the amino acidsequence, Nanobody or polypeptide of the invention) and/or preparativeimmunological techniques (i.e. using antibodies against the amino acidsequence to be isolated).

Generally, for pharmaceutical use, the polypeptides of the invention maybe formulated as a pharmaceutical preparation or compositions comprisingat least one amino acid sequence, Nanobody or polypeptide of theinvention and at least one pharmaceutically acceptable carrier, diluentor excipient and/or adjuvant, and optionally one or more furtherpharmaceutically active polypeptides and/or compounds. By means ofnon-limiting examples, such a formulation may be in a form suitable fororal administration, for parenteral administration (such as byintravenous, intramuscular or subcutaneous injection or intravenousinfusion), for topical administration, for administration by inhalation,by a skin patch, by an implant, by a suppository, etc. Such suitableadministration forms—which may be solid, semi-solid or liquid, dependingon the manner of administration—as well as methods and carriers for usein the preparation thereof, will be clear to the skilled person, and arefurther described herein.

Thus, in a further aspect, the invention relates to a pharmaceuticalcomposition that contains at least one amino acid of the invention, atleast one Nanobody of the invention or at least one polypeptide of theinvention and at least one suitable carrier, diluent or excipient (i.e.suitable for pharmaceutical use), and optionally one or more furtheractive substances. In a particular aspect, the invention relates to apharmaceutical composition that contains SEQ ID NO: 70 and at least onesuitable carrier, diluent or excipient (i.e. suitable for pharmaceuticaluse), and optionally one or more further active substances. In anotherparticular aspect, the invention relates to a pharmaceutical compositionthat contains SEQ ID NO: 71 and at least one suitable carrier, diluentor excipient (i.e. suitable for pharmaceutical use), and optionally oneor more further active substances.

Generally, the amino acid sequences, Nanobodies and polypeptides of theinvention can be formulated and administered in any suitable mannerknown per se, for which reference is for example made to the generalbackground art cited above (and in particular to WO 04/041862, WO04/041863, WO 04/041865 and WO 04/041867) as well as to the standardhandbooks, such as Remington's Pharmaceutical Sciences, le Ed., MackPublishing Company, USA (1990) or Remington, the Science and Practice ofPharmacy, 21th Edition, Lippincott Williams and Wilkins (2005).

For example, the amino acid sequences, Nanobodies and polypeptides ofthe invention may be formulated and administered in any manner known perse for conventional antibodies and antibody fragments (including ScFv'sand diabodies) and other pharmaceutically active proteins. Suchformulations and methods for preparing the same will be clear to theskilled person, and for example include preparations suitable forparenteral administration (for example intravenous, intraperitoneal,subcutaneous, intramuscular, intraluminal, intra-arterial or intrathecaladministration) or for topical (i.e. transdermal or intradermal)administration.

Preparations for parenteral administration may for example be sterilesolutions, suspensions, dispersions or emulsions that are suitable forinfusion or injection. Suitable carriers or diluents for suchpreparations for example include, without limitation, sterile water andaqueous buffers and solutions such as physiological phosphate-bufferedsaline, Ringer's solutions, dextrose solution, and Hank's solution;water oils; glycerol; ethanol; glycols such as propylene glycol or aswell as mineral oils, animal oils and vegetable oils, for example peanutoil, soybean oil, as well as suitable mixtures thereof. Usually, aqueoussolutions or suspensions will be preferred.

The amino acid sequences, Nanobodies and polypeptides of the inventioncan also be administered using gene therapy methods of delivery. See,e.g., U.S. Pat. No. 5,399,346, which is incorporated by reference in itsentirety. Using a gene therapy method of delivery, primary cellstransfected with the gene encoding an amino acid sequence, Nanobody orpolypeptide of the invention can additionally be transfected with tissuespecific promoters to target specific organs, tissue, grafts, tumors, orcells and can additionally be transfected with signal and stabilizationsequences for subcellularly localized expression.

Thus, the amino acid sequences, Nanobodies and polypeptides of theinvention may be systemically administered, e.g., orally, in combinationwith a pharmaceutically acceptable vehicle such as an inert diluent oran assimilable edible carrier. They may be enclosed in hard or softshell gelatin capsules, may be compressed into tablets, or may beincorporated directly with the food of the patient's diet. For oraltherapeutic administration, the amino acid sequences, Nanobodies andpolypeptides of the invention may be combined with one or moreexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.Such compositions and preparations should contain at least 0.1% of theamino acid sequence, Nanobody or polypeptide of the invention. Theirpercentage in the compositions and preparations may, of course, bevaried and may conveniently be between about 2 to about 60% of theweight of a given unit dosage form. The amount of the amino acidsequence, Nanobody or polypeptide of the invention in suchtherapeutically useful compositions is such that an effective dosagelevel will be obtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the amino acid sequences, Nanobodies and polypeptides of theinvention, sucrose or fructose as a sweetening agent, methyl andpropylparabens as preservatives, a dye and flavoring such as cherry ororange flavor. Of course, any material used in preparing any unit dosageform should be pharmaceutically acceptable and substantially non-toxicin the amounts employed. In addition, the amino acid sequences,Nanobodies and polypeptides of the invention may be incorporated intosustained-release preparations and devices.

Preparations and formulations for oral administration may also beprovided with an enteric coating that will allow the constructs of theinvention to resist the gastric environment and pass into theintestines. More generally, preparations and formulations for oraladministration may be suitably formulated for delivery into any desiredpart of the gastrointestinal tract. In addition, suitable suppositoriesmay be used for delivery into the gastrointestinal tract.

The amino acid sequences, Nanobodies and polypeptides of the inventionmay also be administered intravenously or intraperitoneally by infusionor injection. Solutions of the amino acid sequences, Nanobodies andpolypeptides of the invention or their salts can be prepared in water,optionally mixed with a nontoxic surfactant. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, triacetin, andmixtures thereof and in oils. Under ordinary conditions of storage anduse, these preparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form must be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the aminoacid sequences, Nanobodies and polypeptides of the invention in therequired amount in the appropriate solvent with various of the otheringredients enumerated above, as required, followed by filtersterilization. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and the freeze drying techniques, which yield a powder ofthe active ingredient plus any additional desired ingredient present inthe previously sterile-filtered solutions.

For topical administration, the amino acid sequences, Nanobodies andpolypeptides of the invention may be applied in pure form, i.e., whenthey are liquids. However, it will generally be desirable to administerthem to the skin as compositions or formulations, in combination with adermatologically acceptable carrier, which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, hydroxyalkyls or glycols or water-alcohol/glycolblends, in which the amino acid sequences, Nanobodies and polypeptidesof the invention can be dissolved or dispersed at effective levels,optionally with the aid of non-toxic surfactants. Adjuvants such asfragrances and additional antimicrobial agents can be added to optimizethe properties for a given use. The resultant liquid compositions can beapplied from absorbent pads, used to impregnate bandages and otherdressings, or sprayed onto the affected area using pump-type or aerosolsprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Examples of useful dermatological compositions which can be used todeliver the amino acid sequences, Nanobodies and polypeptides of theinvention to the skin are known to the art; for example, see Jacquet etal. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith etal. (U.S. Pat. No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).

Useful dosages of the amino acid sequences, Nanobodies and polypeptidesof the invention can be determined by comparing their in vitro activity,and in vivo activity in animal models. Methods for the extrapolation ofeffective dosages in mice, and other animals, to humans are known to theart; for example, see U.S. Pat. No. 4,938,949.

Generally, the concentration of the amino acid sequence, Nanobodies andpolypeptides of the invention in a liquid composition, such as a lotion,will be from about 0.1-25 wt-%, preferably from about 0.5-10 wt-%. Theconcentration in a semi-solid or solid composition such as a gel or apowder will be about 0.1-5 wt-%, preferably about 0.5-2.5 wt-%.

The amount of the amino acid sequences, Nanobodies and polypeptides ofthe invention required for use in treatment will vary not only with theparticular amino acid sequence, Nanobody or polypeptide selected butalso with the route of administration, the nature of the condition beingtreated and the age and condition of the patient and will be ultimatelyat the discretion of the attendant physician or clinician. Also thedosage of the amino acid sequences, Nanobodies and polypeptides of theinvention varies depending on the target cell, tumor, tissue, graft, ororgan.

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations; such as multiple inhalations from an insufflator or byapplication of a plurality of drops into the eye. An administrationregimen could include long-term, daily treatment. By “long-term” ismeant at least two weeks and preferably, several weeks, months, or yearsof duration. Necessary modifications in this dosage range may bedetermined by one of ordinary skill in the art using only routineexperimentation given the teachings herein. See Remington'sPharmaceutical Sciences (Martin, E. W., ed. 4), Mack Publishing Co.,Easton, Pa. The dosage can also be adjusted by the individual physicianin the event of any complication.

In another aspect, the invention relates to a method for the preventionand/or treatment of at least one IL-6R related disease and/or disorders,said method comprising administering, to a subject in need thereof, apharmaceutically active amount of an amino acid sequence of theinvention, of a Nanobody of the invention, of a polypeptide of theinvention, of a compound of the invention, of a construct of theinvention and/or of a pharmaceutical composition comprising the same.

In the context of the present invention, the term “prevention and/ortreatment” not only comprises preventing and/or treating the disease,but also generally comprises preventing the onset of the disease,slowing or reversing the progress of disease, preventing or slowing theonset of one or more symptoms associated with the disease, reducingand/or alleviating one or more symptoms associated with the disease,reducing the severity and/or the duration of the disease and/or of anysymptoms associated therewith and/or preventing a further increase inthe severity of the disease and/or of any symptoms associated therewith,preventing, reducing or reversing any physiological damage caused by thedisease, and generally any pharmacological action that is beneficial tothe patient being treated.

The subject to be treated may be any warm-blooded animal, but is inparticular a mammal, and more in particular a human being. As will beclear to the skilled person, the subject to be treated will inparticular be a person suffering from, or at risk of, the diseases anddisorders mentioned herein.

The invention relates to a method for the prevention and/or treatment ofat least one disease and/or disorder that is associated with IL-6, withIL-6R, with the IL-6/IL-6R complex, with its biological orpharmacological activity, and/or with the biological pathways orsignalling in which IL-6, IL-6R and/or the IL-6/IL-6R complex areinvolved, said method comprising administering, to a subject in needthereof, a pharmaceutically active amount of an amino acid sequence ofthe invention, of a Nanobody of the invention, of a polypeptide of theinvention, of a compound of the invention, of a construct of theinvention and/or of a pharmaceutical composition comprising the same. Inparticular, the invention relates to a method for the prevention and/ortreatment of at least one disease and/or disorder that can be preventedand/or treated by modulating IL-6, IL-6R, the IL-6/IL-6R complex, itsbiological or pharmacological activity, and/or the biological pathwaysor signalling in which IL-6, IL-6R and/or the IL-6/IL-6R complex isinvolved, said method comprising administering, to a subject in needthereof, a pharmaceutically active amount of an amino acid sequence ofthe invention, of a Nanobody of the invention, of a polypeptide, of acompound of the invention, of a construct of the invention of theinvention, and/or of a pharmaceutical composition comprising the same.In particular, said pharmaceutically effective amount may be an amountthat is sufficient to modulate IL-6, IL-6R, the EL-6/IL-611 complex, itsbiological or pharmacological activity, and/or the biological pathwaysor signalling in which IL-6. IL-6R and/or the IL-6/IL-6R complex areinvolved.

The invention also relates to a method for the prevention and/ortreatment of at least one disease and/or disorder that can be preventedand/or treated by administering of an amino acid sequence of theinvention, a Nanobody of the invention or a polypeptide of the inventionto a patient, said method comprising administering, to a subject in needthereof, a pharmaceutically active amount of an amino acid sequence ofthe invention, of a Nanobody of the invention, of a polypeptide of theinvention, of a compound of the invention, of a construct of theinvention and/or of a pharmaceutical composition comprising the same.

More in particular, the invention relates to a method for the preventionand/or treatment of at least one disease and/or disorder chosen from thegroup consisting of the diseases and disorders listed herein, saidmethod comprising administering, to a subject in need thereof, apharmaceutically active amount of an amino acid sequence of theinvention, of a Nanobody of the invention, of a polypeptide of theinvention, of a compound of the invention, of a construct of theinvention and/or of a pharmaceutical composition comprising the same.

In particular, the present invention relates to a method for theprevention and/or treatment of sepsis, various forms of cancer, boneresorption, osteoporosis, cachexia, psoriasis, mesangial proliferativeglomerulonephritis, Kaposi's sarcoma, AIDS-related lymphoma, andinflammatory diseases, said method comprising administering apharmaceutically active amount of an amino acid sequence of theinvention, of a Nanobody of the invention, of a polypeptide of theinvention, and/or of a pharmaceutical composition comprising the same.The various forms of cancer may be chosen from the group consisting ofmultiple myeloma disease (MM), renal cell carcinoma (RCC), plasma cellleukaemia, lymphoma, B-lymphoproliferative disorder (BLPD), and prostatecancer. The inflammatory diseases may be chosen from the groupconsisting of rheumatoid arthritis, systemic onset juvenile idiopathicarthritis, hypergammaglobulinemia, Crohn's disease, ulcerative colitis,systemic lupus erythematosus (SLE), multiple sclerosis, Castleman'sdisease, IgM gammopathy, cardiac myxoma, asthma, allergic asthma andautoimmune insulin-dependent diabetes mellitus.

In another particular aspect, the present invention relates to a methodfor the prevention and/or treatment of sepsis, various forms of cancer,bone resorption, osteoporosis, cachexia, psoriasis, mesangialproliferative glomerulonephritis, Kaposi's sarcoma, AIDS-relatedlymphoma, and inflammatory diseases, said method comprisingadministering a pharmaceutically active amount of SEQ ID NO: 70, and/orof a pharmaceutical composition comprising the same. In anotherparticular aspect, the present invention relates to a method for theprevention and/or treatment of sepsis, various forms of cancer, boneresorption, osteoporosis, cachexia, psoriasis, mesangial proliferativeglomerulonephritis, Kaposi's sarcoma, AIDS-related lymphoma, andinflammatory diseases, said method comprising administering apharmaceutically active amount of SEQ ID NO: 71, and/or of apharmaceutical composition comprising the same. The various forms ofcancer may be chosen from the group consisting of multiple myelomadisease (MM), renal cell carcinoma (RCC), plasma cell leukaemia,lymphoma, B-lymphoproliferative disorder (BLPD), and prostate cancer.The inflammatory diseases may be chosen from the group consisting ofrheumatoid arthritis, systemic onset juvenile idiopathic arthritis,hypergammaglobulinemia, Crohn's disease, ulcerative colitis, systemiclupus erythematosus (SLE), multiple sclerosis, Castleman's disease, IgMgammopathy, cardiac myxoma, asthma, allergic asthma and autoimmuneinsulin-dependent diabetes mellitus.

In another embodiment, the invention relates to a method forimmunotherapy, and in particular for passive immunotherapy, which methodcomprises administering, to a subject suffering from or at risk of thediseases and disorders mentioned herein, a pharmaceutically activeamount of an amino acid sequence of the invention, of a Nanobody of theinvention, of a polypeptide of the invention, of a compound of theinvention, of a construct of the invention and/or of a pharmaceuticalcomposition comprising the same.

In the above methods, the amino acid sequences, Nanobodies,polypeptides, compounds and/or constructs of the invention and/or thecompositions comprising the same can be administered in any suitablemanner, depending on the specific pharmaceutical formulation orcomposition to be used. Thus, the amino acid sequences, Nanobodies,polypeptides, compounds and/or constructs of the invention and/or thecompositions comprising the same can for example be administered orally,intraperitoneally (e.g. intravenously, subcutaneously, intramuscularly,or via any other route of administration that circumvents thegastrointestinal tract), intranasally, transdermally, topically, bymeans of a suppository, by inhalation, again depending on the specificpharmaceutical formulation or composition to be used. The clinician willbe able to select a suitable route of administration and a suitablepharmaceutical formulation or composition to be used in suchadministration, depending on the disease and/or disorder to be preventedor treated and other factors well known to the clinician.

The amino acid sequences, Nanobodies, polypeptides, compounds and/orconstructs of the invention and/or the compositions comprising the sameare administered according to a regime of treatment that is suitable forpreventing and/or treating the disease and/or disorder to be preventedor treated. The clinician will generally be able to determine a suitabletreatment regimen, depending on factors such as the disease or disorderto be prevented or treated, the severity of the disease to be treatedand/or the severity of the symptoms thereof, the specific amino acidsequences, Nanobody, polypeptide, compound or construct of the inventionto be used, the specific route of administration and pharmaceuticalformulation or composition to be used, the age, gender, weight, diet,general condition of the patient, and similar factors well known to theclinician.

Generally, the treatment regimen will comprise the administration of oneor more amino acid sequences, Nanobodies, polypeptides, compounds and/orconstructs of the invention, or of one or more compositions comprisingthe same, in one or more pharmaceutically effective amounts or doses.The specific amount(s) or doses to administered can be determined by theclinician, again based on the factors cited above.

Generally, for the prevention and/or treatment of the diseases anddisorders mentioned herein and depending on the specific disease ordisorder to be treated, the potency of the specific amino acid sequence,Nanobody, polypeptide, compound and construct of the invention to beused, the specific route of administration and the specificpharmaceutical formulation or composition used, the amino acidsequences, Nanobodies, polypeptides, compounds and constructs of theinvention will generally be administered in an amount between 1 gram and0.01 microgram per kg body weight per day, preferably between 0.1 gramand 0.1 microgram per kg body weight per day, such as about 1, 10, 100or 1000 microgram per kg body weight per day, either continuously (e.g.by infusion), as a single daily dose or as multiple divided doses duringthe day. The clinician will generally be able to determine a suitabledaily dose, depending on the factors mentioned herein. It will also beclear that in specific cases, the clinician may choose to deviate fromthese amounts, for example on the basis of the factors cited above andhis expert judgment. Generally, some guidance on the amounts to beadministered can be obtained from the amounts usually administered forcomparable conventional antibodies or antibody fragments against thesame target administered via essentially the same route, taking intoaccount however differences in affinity/avidity, efficacy,biodistribution, and similar factors well known to the skilled person.

Usually, in the above method, a single amino acid sequence, Nanobody,polypeptide, compound or construct of the invention will be used. It ishowever within the scope of the invention to use two or more amino acidsequences, Nanobodies, polypeptides, compounds and/or constructs of theinvention in combination.

The amino acid sequences, Nanobodies, polypeptides, compounds andconstructs of the invention may also be used in combination with one ormore further pharmaceutically active compounds or principles, i.e. as acombined treatment regimen, which may or may not lead to a synergisticeffect. Again, the clinician will be able to select such furthercompounds or principles, as well as a suitable combined treatmentregimen, based on the factors cited above and his expert judgement.

In particular, the amino acid sequences, Nanobodies, polypeptides,compounds and constructs of the invention may be used in combinationwith other pharmaceutically active compounds or principles that are orcan be used for the prevention and/or treatment of the diseases anddisorders cited herein, as a result of which a synergistic effect may ormay not be obtained. Examples of such compounds and principles, as wellas routes, methods and pharmaceutical formulations or compositions foradministering them will be clear to the clinician.

When two or more substances or principles are to be used as part of acombined treatment regimen, they can be administered via the same routeof administration or via different routes of administration, atessentially the same time or at different times (e.g. essentiallysimultaneously, consecutively, or according to an alternating regime).When the substances or principles are to be administered simultaneouslyvia the same route of administration, they may be administered asdifferent pharmaceutical formulations or compositions or part of acombined pharmaceutical formulation or composition, as will be clear tothe skilled person.

Also, when two or more active substances or principles are to be used aspart of a combined treatment regimen, each of the substances orprinciples may be administered in the same amount and according to thesame regimen as used when the compound or principle is used on its own,and such combined use may or may not lead to a synergistic effect.However, when the combined use of the two or more active substances orprinciples leads to a synergistic effect, it may also be possible toreduce the amount of one, more or all of the substances or principles tobe administered, while still achieving the desired therapeutic action.This may for example be useful for avoiding, limiting or reducing anyunwanted side-effects that are associated with the use of one or more ofthe substances or principles when they are used in their usual amounts,while still obtaining the desired pharmaceutical or therapeutic effect.

The effectiveness of the treatment regimen used according to theinvention may be determined and/or followed in any manner known per sefor the disease and/or disorder involved, as will be clear to theclinician. The clinician will also be able, where appropriate and on acase-by-case basis, to change or modify a particular treatment regimen,so as to achieve the desired therapeutic effect, to avoid, limit orreduce unwanted side-effects, and/or to achieve an appropriate balancebetween achieving the desired therapeutic effect on the one hand andavoiding, limiting or reducing undesired side effects on the other hand.

Generally, the treatment regimen will be followed until the desiredtherapeutic effect is achieved and/or for as long as the desiredtherapeutic effect is to be maintained. Again, this can be determined bythe clinician.

In another aspect, the invention relates to the use of an amino acidsequence, Nanobody, polypeptide, compound or (monovalent) construct ofthe invention in the preparation of a pharmaceutical composition forprevention and/or treatment of at least one IL-6R related disorders.

The invention also relates to the use of an amino acid sequence,Nanobody, polypeptide, compound or (monovalent) construct of theinvention, in the preparation of a pharmaceutical composition forprevention and/or treatment of at least one of the diseases anddisorders associated with IL-6, with IL-6R, with theft-6/IL-6R complexand/or with the signalling pathways and/or the biological functions andresponses in which IL-6, IL-6R and/or the IL-6/IL-6R complex areinvolved; and/or for use in one or more of the methods described herein.

The invention also relates to the use of an amino acid sequence,Nanobody, polypeptide, compound or construct of the invention in thepreparation of a pharmaceutical composition for the prevention and/ortreatment of at least one disease or disorder that can be preventedand/or treated by modulating IL-6, IL-6R, the IL-6/IL-6R complex, itsbiological or pharmacological activity, and/or the biological pathwaysor signalling in which IL-6, IL-6R and/or the IL-6/IL-6R complex isinvolved.

The invention also relates to the use of an amino acid sequence,Nanobody, polypeptide, compound or construct of the invention in thepreparation of a pharmaceutical composition for the prevention and/ortreatment of at least one disease or disorder that can be preventedand/or treated by administering an amino acid sequence, Nanobody,polypeptide, compound or construct of the invention to a patient.

More in particular, the invention relates to the use of an amino acidsequence, Nanobody, polypeptide, compound or construct of the inventionin the preparation of a pharmaceutical composition for the preventionand/or treatment of IL-6R related disorders, and in particular for theprevention and treatment of a sepsis, various forms of cancer, boneresorption, osteoporosis, cachexia, psoriasis, mesangial proliferativeglomerulonephritis, Kaposi's sarcoma, AIDS-related lymphoma, andinflammatory diseases, said method comprising administering apharmaceutically active amount of an amino acid sequence of theinvention, of a Nanobody of the invention, of a polypeptide of theinvention, and/or of a pharmaceutical composition comprising the same.The various forms of cancer may be chosen from the group consisting ofmultiple myeloma disease (MM), renal cell carcinoma (RCC), plasma cellleukaemia, lymphoma, B-lymphoproliferative disorder (BLPD), and prostatecancer. The inflammatory diseases may be chosen from the groupconsisting of rheumatoid arthritis, systemic onset juvenile idiopathicarthritis, hypergammaglobulinemia, Crohn's disease, ulcerative colitis,systemic lupus erythematosus (SLE), multiple sclerosis, Castleman'sdisease, IgM gammopathy, cardiac myxoma, asthma, allergic asthma andautoimmune insulin-dependent diabetes mellitus.

The invention further relates to an amino acid sequence, a Nanobody, acompound or construct, a polypeptide, monovalent construct of theinvention or a pharmaceutical composition comprising the same for use inthe prevention and/or treatment of at least one IL-6R related diseaseand/or disorder.

The invention further relates to an amino acid sequence, a Nanobody, acompound or construct, a polypeptide, monovalent construct of theinvention or a pharmaceutical composition comprising the same for use inthe prevention and/or treatment of at least one disease and/or disorderassociated with IL-6, with IL-6R, with the IL-6/IL-6R complex, with itsbiological or pharmacological activity, and/or with the biologicalpathways or signalling in which IL-6, IL-6R and/or the IL-6/IL-6Rcomplex are involved.

The invention further relates to an amino acid sequence, a Nanobody, acompound or construct, a polypeptide, monovalent construct of theinvention or a pharmaceutical composition comprising the same for use inthe prevention and/or treatment of at least one disease and/or disorderthat can be prevented and/or treated by modulating IL-6, IL-6R, theIL-6/IL-6R complex, its biological or pharmacological activity, and/orthe biological pathways or signalling in which IL-6, IL-6R and/or theIL-6/IL-6R complex is involved.

The invention further relates to an amino acid sequence, a Nanobody, acompound or construct, a polypeptide, monovalent construct of theinvention or a pharmaceutical composition comprising the same for use inthe prevention and/or treatment of at least one disease and/or disorderthat can be prevented and/or treated by administering of an amino acidsequence of the invention, a Nanobody of the invention or a polypeptideof the invention to a patient.

The invention further relates to an amino acid sequence, a Nanobody, acompound or construct, a polypeptide, monovalent construct of theinvention or a pharmaceutical composition comprising the same for use inthe prevention and/or treatment of sepsis, various forms of cancer, boneresorption, osteoporosis, cachexia, psoriasis, mesangial proliferativeglomerulonephritis, Kaposi's sarcoma, AIDS-related lymphoma, andinflammatory diseases, said method comprising administering apharmaceutically active amount of an amino acid sequence of theinvention, of a Nanobody of the invention, of a polypeptide of theinvention, and/or of a pharmaceutical composition comprising the same.The various forms of cancer may be chosen from the group consisting ofmultiple myeloma disease (MM), renal cell carcinoma (RCC), plasma cellleukaemia, lymphoma, B-lymphoproliferative disorder (BLPD), and prostatecancer. The inflammatory diseases may be chosen from the groupconsisting of rheumatoid arthritis, systemic onset juvenile idiopathicarthritis, hypergammaglobulinemia, Crohn's disease, ulcerative colitis,systemic lupus erythematosus (SLE), multiple sclerosis, Castleman'sdisease, IgM gammopathy, cardiac myxoma, asthma, allergic asthma andautoimmune insulin-dependent diabetes mellitus.

The subject to be treated may be any warm-blooded animal, but is inparticular a mammal, and more in particular a human being. As will beclear to the skilled person, the subject to be treated will inparticular be a person suffering from, or at risk of, the diseases anddisorders mentioned herein.

Again, in such a pharmaceutical composition, the one or more amino acidsequences, Nanobodies, polypeptides, compounds or constructs of theinvention may also be suitably combined with one or more other activeprinciples, such as those mentioned herein.

The present invention is further illustrated by the following Examples,which in no way should be construed as further limiting. The entirecontents of all of the references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference, in particular for the teaching that isreferenced hereinabove.

Aspects

-   Aspect 1. Amino acid sequence directed against IL-6R, that comprises    one or more stretches of amino acid residues chosen from the    following:    -   a) SEQ ID NO's: 80-82; or    -   b) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with one of SEQ        ID NO's: 80-82, provided that the amino acid sequence comprising        said stretch of amino acid residues binds IL-6R with about the        same or a higher affinity compared to the amino acid sequence        comprising said stretch of amino acid residues without the 2 or        1 amino acid difference, said affinity as measured by surface        plasmon resonance;    -   and/or    -   c) SEQ ID NO's: 84-91; or    -   d) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with one of SEQ        ID NO's: 84-91, provided that the amino acid sequence comprising        said stretch of amino acid residues binds IL-6R with about the        same or a higher affinity compared to the amino acid sequence        comprising said stretch of amino acid residues without the 2 or        1 amino acid difference, said affinity as measured by surface        plasmon resonance;    -   and/or    -   e) SEQ ID NO's: 93-95; or    -   f) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with one of SEQ        ID NO's: 93-95, provided that the amino acid sequence comprising        said stretch of amino acid residues binds IL-6R with about the        same or a higher affinity compared to the amino acid sequence        comprising said stretch of amino acid residues without the 1 or        2 amino add difference, said affinity as measured by surface        plasmon resonance.-   Aspect 2. Amino acid sequence according to aspect 1, that comprises    two or more stretches of amino acid residues chosen from the    following:    -   a) SEQ ID NO's: 80-82; or    -   b) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with one of SEQ        ID NO's: 80-82, provided that the amino acid sequence comprising        said stretch of amino acid residues binds IL-6R with about the        same or a higher affinity compared to the amino acid sequence        comprising said stretch of amino acid residues without the 2 or        1 amino acid difference, said affinity as measured by surface        plasmon resonance;    -   and/or    -   c) SEQ ID NO's: 84-91; or    -   d) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with one of SEQ        ID NO's: 84-91, provided that the amino acid sequence comprising        said stretch of amino acid residues binds IL-6R with about the        same or a higher affinity compared to the amino acid sequence        comprising said stretch of amino acid residues without the 2 or        1 amino acid difference, said affinity as measured by surface        plasmon resonance;    -   and/or    -   e) SEQ ID NO's: 93-95; or    -   f) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with one of SEQ        ID NO's: 93-95, provided that the amino acid sequence comprising        said stretch of amino acid residues binds IL-6R with about the        same or a higher affinity compared to the amino acid sequence        comprising said stretch of amino acid residues without the 1 or        2 amino acid difference, said affinity as measured by surface        plasmon resonance.    -   such that (i) when the first stretch of amino acid residues        corresponds to one of the amino acid sequences according to a),        or b), the second stretch of amino acid residues corresponds to        one of the amino acid sequences according to c), d), e) or        f); (ii) when the first stretch of amino acid residues        corresponds to one of the amino acid sequences according to c)        or d), the second stretch of amino acid residues corresponds to        one of the amino acid sequences according to a), b), e) or f);        or (iii) when the first stretch of amino acid residues        corresponds to one of the amino acid sequences according to e)        or f), the second stretch of amino acid residues corresponds to        one of the amino acid sequences according to a), b), c) or d).-   Aspect 3. Amino acid sequence according to any of aspects 1 or 2,    that comprises three or more stretches of amino acid residues, in    which the first stretch of amino acid residues is chosen from the    group consisting of:    -   a) SEQ ID NO's: 80-82; or    -   b) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with one of SEQ        ID NO's: 80-82, provided that the amino acid sequence comprising        said stretch of amino acid residues binds IL-6R with about the        same or a higher affinity compared to the amino acid sequence        comprising said stretch of amino acid residues without the 2 or        1 amino acid difference, said affinity as measured by surface        plasmon resonance;    -   the second stretch of amino acid residues is chosen from the        group consisting of:    -   c) SEQ ID NO's: 84-91; or    -   d) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with one of SEQ        ID ND's: 84-91, provided that the amino acid sequence comprising        said stretch of amino acid residues binds IL-6R with about the        same or a higher affinity compared to the amino acid sequence        comprising said stretch of amino acid residues without the 2 or        1 amino acid difference, said affinity as measured by surface        plasmon resonance;    -   and the third stretch of amino acid residues is chosen from the        group consisting of:    -   e) SEQ ID ND's; 93-95; or    -   f) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with one of SEQ        ID NO's: 93-95, provided that the amino acid sequence comprising        said stretch of amino acid residues binds IL-6R with about the        same or a higher affinity compared to the amino acid sequence        comprising said stretch of amino acid residues without the 1 or        2 amino acid difference, said affinity as measured by surface        plasmon resonance.-   Aspect 4. Amino acid sequence according to any of aspects 1 to 3,    which comprises an immunoglobulin fold or which under suitable    conditions is capable of forming an immunoglobulin fold,-   Aspect 5. Amino acid sequence according to any of aspects 1 to 4,    which is an immunoglobulin sequence.-   Aspect 6. Amino acid sequence according to any of aspects 1 to 5,    which essentially consists of 4 framework regions (FR1 to FR4,    respectively) and 3 complementarity determining regions (CDR1 to    CDR3, respectively), in which:    -   CDR1 is chosen from the group consisting of:    -   a) SEQ ID NO's: 80-82; or    -   b) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with one of SEQ        ID NO's: 80-82, provided that the amino acid sequence comprising        said stretch of amino acid residues binds IL-6R with about the        same or a higher affinity compared to the amino acid sequence        comprising said stretch of amino acid residues without the 2 or        1 amino acid difference, said affinity as measured by surface        plasmon resonance;    -   and/or    -   CDR2 is chosen from the group consisting of:    -   c) SEQ ID NO's: 84-91; or    -   d) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with one of SEQ        ID NO's: 84-91, provided that the amino acid sequence comprising        said stretch of amino acid residues binds IL-6R with about the        same or a higher affinity compared to the amino acid sequence        comprising said stretch of amino acid residues without the 2 or        1 amino acid difference, said affinity as measured by surface        plasmon resonance;    -   and/or    -   CDR3 is chosen from the group consisting of:    -   e) SEQ ID NO's: 93-95; or    -   f) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with one of SEQ        ID NO's: 93-95, provided that the amino acid sequence comprising        said stretch of amino acid residues binds IL-6R with about the        same or a higher affinity compared to the amino acid sequence        comprising said stretch of amino acid residues without the 1 or        2 amino acid difference, said affinity as measured by surface        plasmon resonance.-   Aspect 7. Amino acid sequence according to aspect 6, which    essentially consists of 4 framework regions (FR1 to FR4,    respectively) and 3 complementarity determining regions (CDR1 to    CDR3, respectively), in which:    -   CDR1 is chosen from the group consisting of:    -   a) SEQ ID NO's: 80-82; or    -   b) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with one of SEQ        ID NO's: 80-82, provided that the amino acid sequence comprising        said stretch of amino acid residues binds IL-6R with about the        same or a higher affinity compared to the amino acid sequence        comprising said stretch of amino acid residues without the 2 or        1 amino acid difference, said affinity as measured by surface        plasmon resonance;    -   and    -   CDR2 is chosen from the group consisting of:    -   c) SEQ ID NO's: 84-91; or    -   d) a stretch of amino add residues that has no more than 2,        preferably no more than 1 amino acid difference with one of SEQ        ID NO's: 84-91, provided that the amino acid sequence comprising        said stretch of amino acid residues binds IL-6R with about the        same or a higher affinity compared to the amino acid sequence        comprising said stretch of amino acid residues without the 2 or        1 amino acid difference, said affinity as measured by surface        plasmon resonance;    -   and    -   CDR3 is chosen from the group consisting of:    -   e) SEQ ID NO's: 93-95; or    -   f) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with one of SEQ        ID NO's: 93-95, provided that the amino acid sequence comprising        said stretch of amino acid residues binds IL-6R with about the        same or a higher affinity compared to the amino acid sequence        comprising said stretch of amino acid residues without the 1 or        2 amino acid difference, said affinity as measured by surface        plasmon resonance.-   Aspect 8. Amino acid sequence according to any of aspects 1 to 7,    which comprises at least:    -   a) SEQ ID NO: 80; or    -   b) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with SEQ ID NO:        80, provided that the amino acid sequence comprising said        stretch of amino acid residues binds IL-6R with about the same        or a higher affinity compared to the amino acid sequence        comprising said stretch of amino acid residues without the 2 or        1 amino acid difference, said affinity as measured by surface        plasmon resonance.-   Aspect 9. Amino acid sequence according to any of aspects 1 to 8,    which comprises at least a stretch of amino acid residues chosen    from the following:    -   a) SEQ ID NO's: 84, 89 or 91; or    -   b) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with one of SEQ        ID NO's: 84, 89 or 91, provided that the amino acid sequence        comprising said stretch of amino acid residues binds IL-6R with        about the same or a higher affinity compared to the amino acid        sequence comprising said stretch of amino acid residues without        the 1 or 2 amino acid difference, said affinity as measured by        surface plasmon resonance.-   Aspect 10. Amino acid sequence according to any of aspects 1 to 9,    which comprises at least:    -   a) SEQ ID NO: 84; or    -   b) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with SEQ ID NO:        84, provided that the amino acid sequence comprising said        stretch of amino acid residues binds IL-6R with about the same        or a higher affinity compared to the amino acid sequence        comprising said stretch of amino acid residues without the 2 or        1 amino acid difference, said affinity as measured by surface        plasmon resonance.-   Aspect 11. Amino acid sequence according to any of aspects 1 to 10,    which comprises at least a stretch of amino acid residues chosen    from the following:    -   a) SEQ ID NO's: 93-94; or    -   b) a stretch of amino acid residues that has no more than 2,        preferably no more than 1 amino acid difference with one of SEQ        ID NO's: 93-94, provided that the amino acid sequence comprising        said stretch of amino acid residues binds IL-6R with about the        same or a higher affinity compared to the amino acid sequence        comprising said stretch of amino acid residues without the 1 or        2 amino acid difference, said affinity as measured by surface        plasmon resonance.-   Aspect 12. Amino acid sequence according to any of aspects 1 to 11,    which comprises at least:    -   a) SEQ ID NO: 93; or    -   b) a stretch of amino acid residues that has no more than 2,        preferably no more than amino acid difference with SEQ ID NO:        93, provided that the amino acid sequence comprising said        stretch of amino acid residues binds IL-6R with about the same        or a higher affinity compared to the amino acid sequence        comprising said stretch of amino acid residues without the 2 or        1 amino acid difference, said affinity as measured by surface        plasmon resonance.-   Aspect 13. Amino acid sequence according to any of aspects 1 to 12,    which comprises at least two stretches of amino acid residues    selected from:    -   a) SEQ ID NO: 80 and SEQ ID NO: 84;    -   b) SEQ ID NO: 80 and SEQ ID NO: 93; or    -   c) SEQ ID NO: 84 and SEQ ID NO: 93.-   Aspect 14. Amino acid sequence according to any of aspects 1 to 13,    which comprises SEQ ID NO: 80, SEQ ID NO: 84 and SEQ ID NO: 93.-   Aspect 15. Amino acid sequence according to any of aspects 1 to 14,    which essentially consists of a heavy chain variable domain sequence    that is derived from a conventional four-chain antibody or which    essentially consist of a heavy chain variable domain sequence that    is derived from heavy chain antibody.-   Aspect 16. Amino acid sequence according to any of aspects 1 to 15,    which essentially consists of a domain antibody (or an amino acid    sequence that is suitable for use as a domain antibody), of a single    domain antibody (or an amino acid sequence that is suitable for use    as a single domain antibody), of a “dAb” (or an amino acid sequence    that is suitable for use as a dAb) or of a Nanobody.-   Aspect 17. Amino acid sequence according to any of aspects 1 to 16    selected from the group consisting of:    -   a) SEQ ID NO's: 60-69;    -   b) a sequence that has no more than 2, preferably no more than 1        amino acid difference in one, two or all of its CDRs with one of        SEQ ID NO's: 60-69, provided that the amino acid sequence with        no more than 2, preferably no more than 1 amino acid difference        in one, two or all of its CDRs binds IL-6R with about the same        or a higher affinity compared to the binding by the one of SEQ        ID NO's: 60-69, said affinity as measured by surface plasmon        resonance;    -   c) a sequence that has no more than 2, preferably no more than 1        amino acid difference with one of SEQ ID NO's: 60-69, provided        that the amino acid sequence with no more than 2, preferably no        more than 1 amino acid difference with one of SEQ ID NO's: 50-69        binds IL-6R with about the same or a higher affinity compared to        the binding by the one of SEQ ID NO's: 60-69, said affinity as        measured by surface plasmon resonance.-   Aspect 18. Amino acid sequence according to aspect 17 selected from    the group consisting of:    -   a) SEQ ID NO's: 65-69;    -   b) a sequence that has no more than 2, preferably no more than 1        amino acid difference in one, two or all of its CDRs with one of        SEQ ID NO's: 65-69, provided that the amino acid sequence with        no more than 2, preferably no more than 1 amino acid difference        in one, two or all of its CDRs binds IL-6R with about the same        or a higher affinity compared to the binding by the one of SEQ        ID NO's: 65-69, said affinity as measured by surface plasmon        resonance;    -   c) a sequence that has no more than 2, preferably no more than 1        amino acid difference with one of SEQ ID NO's: 65-69, provided        that the amino acid sequence with no more than 2, preferably no        more than 1 amino acid difference with one of SEQ ID NO's: 65-69        binds IL-6R with about the same or a higher affinity compared to        the binding by the one of SEQ ID NO's: 65-69, said affinity as        measured by surface plasmon resonance.-   Aspect 19. Amino acid sequence according to aspect 18 selected from    the group consisting of:    -   a) SEQ ID NO: 66;    -   b) a sequence that has no more than 2, preferably no more than 1        amino acid difference in one, two or all of its CDRs with SEQ ID        NO: 66, provided that the amino acid sequence with no more than        2, preferably no more than 1 amino acid difference in one, two        or all of its CDRs binds IL-6R with about the same or a higher        affinity compared to the binding by SEQ ID NO: 66, said affinity        as measured by surface plasmon resonance;    -   c) a sequence that has no more than 2, preferably no more than 1        amino acid difference with SEQ ID NO: 66, provided that the        amino acid sequence with no more than 2, preferably no more than        1 amino acid difference with SEQ ID NO: 66 binds IL-6R with        about the same or a higher affinity compared to the binding by        SEQ ID NO:66, said affinity as measured by surface plasmon        resonance.-   Aspect 20. Amino acid sequence according to any of aspects 1 to 19,    that specifically binds to hIL-6R with a dissociation constant    (K_(D)) of 1 nM to 1 pM moles/litre or less, preferably 500 pM to 1    pM moles/litre or less, more preferably 100 pM to 1 pM moles/litre    or less, or even more preferably about 50 pM to 1 pM or less.-   Aspect 21. Amino acid sequence according to any of aspects 1 to 20,    that specifically binds to cyno IL-6R with a dissociation constant    (K_(D)) of 1 nM to 1 pM or less, preferably 500 pM to 1 pM or less,    more preferably 100 pM to 1 pM or less, or even more preferably    about 50 pM to 1 pM or less.-   Aspect 22. Amino acid sequence according to any of aspects 1 to 21,    that specifically binds to hIL-6R with a k_(on)-rate of between 10⁴    M⁻¹s⁻¹ to about 10⁷ M⁻¹s⁻¹, preferably between 10⁵ M⁻¹s⁻¹ and 10⁷    M⁻¹ s⁻¹, more preferably about 10⁶ M⁻¹s⁻¹ or more.-   Aspect 23. Amino acid sequence according to any of aspects 1 to 22,    that specifically binds to cyno IL-6R with a k_(on)-rate of between    10⁴ M⁻¹s⁻¹ to about 10⁷ M⁻¹s⁻¹, preferably between 10⁵ M⁻¹s⁻¹ and    10⁷ M⁻¹s⁻¹, more preferably about 10⁶ M⁻¹s⁻¹ or more.-   Aspect 24. Amino acid sequence according to any of aspects 1 to 23,    that specifically binds to hIL-6R with a k_(off) rate between 10⁻³    s⁻¹ (t_(1/2)=0.69 s) and 10⁻⁶ s⁻¹ (providing a near irreversible    complex with a t_(1/2) of multiple days), preferably between 10⁻⁴    s⁻¹ and 10⁻⁶ s⁻¹, more preferably between 10⁻⁵ s⁻¹ and 10⁻⁶ s⁻¹,    such as about 10⁻⁵ s⁻¹ or lower.-   Aspect 25. Amino acid sequence according to any of aspects 1 to 24,    that specifically binds to cyno IL-6R with a k_(off) rate between    10⁻³ s⁻¹ (t_(1/2)=0.69 s) and 10⁻⁶ s⁻¹ (providing a near    irreversible complex with a t_(1/2) of multiple days), preferably    between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹, more preferably between 10⁻⁵ s⁻¹ and    10⁻⁶ s⁻¹, such as about 10⁻⁶ s⁻¹ or lower.-   Aspect 26. Amino acid sequence according to any of aspects 1 to 25,    that, in the TF-1 assay, has IC50 values (at 100 IU/mL IL-6) between    10 nM and 50 pM, preferably between 5 nM and 50 pM, more preferably    between 1 nM and 50 pM or less, such as about 750 or 500 pM or less.-   Aspect 27. Amino acid sequence according to any of aspects 1 to 26,    that, in the TF-1 assay, has IC50 (at 5000 ILD/mL IL-6) between 50    nM and 1 nM, preferably between 25 nM and 1 nM, more preferably    between 10 nM and 1 nM or less, such as about 8 nM or less.-   Aspect 28. Amino acid sequence according to any of aspects 1 to 27,    that, in the TF-1 assay, has IC50 values that are at least the same    and preferably better, at least two times, preferably three times,    more preferably four times, even more preferably 5 times, 7 times or    more than 7 times better compared to the IC50 value obtained for the    reference IgG as defined by SEQ ID NO's: 1 and 2 or the reference    Fab as defined by SEQ ID NO's: 3 and 4-   Aspect 29. Amino acid sequence according to any of aspects 1 to 28,    that, in the TF-1 assay, has IC50 values that are at least the same    and preferably better, at least two times, preferably three times,    more preferably four times, even more preferably 5 times, 7 times or    more than 7 times better compared to the IC50 value obtained for    Tocilizumab (MRA).-   Aspect 30. Amino acid sequence according to any of aspects 1 to 29,    that has, in a plasma potency assay at EC50 values of IL-6, IC50    values between 500 pM and 50 pM, preferably between 250 pM and 50    pM, more preferably between 200 pM and 50 pM or less, such as 150 pM    or less.-   Aspect 31. Amino acid sequence according to any of aspects 1 to 30,    that has, in a plasma potency assay at EC95 values of IL-6, IC50    values between 1000 pM and 100 pM, preferably between 750 pM and 100    pM, more preferably between 500 pM and 100 pM or less, such as 400    pM or less.-   Aspect 32. Amino acid sequence according to any of aspects 1 to 31,    that has, in a plasma potency assay of aspects 30 or 31, IC50 values    that are at least the same and preferably better, at least two    times, preferably three times, more preferably four times, even more    preferably 5 times, 7 times or more than 7 times better compared to    the IC50 value obtained for the reference IgG as defined by SEQ ID    NO's: 1 and 2 or the reference Fab as defined by SEQ ID NO's: 3 and    4.-   Aspect 33. Amino acid sequence according to any of aspects 1 to 32,    that has, in a plasma potency assay of aspects 30 or 31, IC50 values    that are at least the same and preferably better, at least two    times, preferably three times, more preferably four times, even more    preferably 5 times, 7 times or more than 7 times better compared to    the IC50 value obtained for Tocilizumab (MRA).-   Aspect 34. Amino acid sequence according to any of aspects 1 to 33,    that has, for binding to membrane IL-6R on CHO cells, IC50 values    between 10 nM and 100 pM, preferably between 5 nM and 100 pM, more    preferably between 2 nM and 10 pM or less, such as 2 nM or less.-   Aspect 35. Compound or construct, that comprises or essentially    consists of one or more amino acid sequences according to any of    aspects 1 to 34, and optionally further comprises one or more other    groups, residues, moieties or binding units, optionally linked via    one or more linkers.-   Aspect 36. Compound or construct according to aspect 35, in which    said one or more other groups, residues, moieties or binding units    are chosen from the group consisting of domain antibodies, amino    acid sequences that are suitable for use as a domain antibody,    single domain antibodies, amino acid sequences that are suitable for    use as a single domain antibody, “dAb”'s, amino acid sequences that    are suitable for use as a dAb, or Nanobodies,-   Aspect 37. Compound or construct according to any of aspects 35 or    36, which is a multivalent construct, such as e.g. a bivalent or    trivalent construct.-   Aspect 38. Compound or construct according to any of aspects 35 to    37, which is a multispecific construct, such as e.g. a bispecific or    trispecific construct.-   Aspect 39. Compound or construct according to any of aspects 35 to    38, which has an increased half-life, compared to the corresponding    amino acid sequence according to any of aspects 1 to 34 per se.-   Aspect 40. Compound or construct according to aspect 39, in which    said one or more other groups, residues, moieties or binding units    provide the compound or construct with increased half-life, compared    to the corresponding amino acid sequence according to any of aspects    1 to 20.-   Aspect 41. Compound or construct according to aspect 39, in which    said one or more other groups, residues, moieties or binding units    that provide the compound or construct with increased half-life is    chosen from the group consisting of serum proteins or fragments    thereof, binding units that can bind to serum proteins, an Pc    portion, and small proteins or peptides that can bind to serum    proteins.-   Aspect 42. Compound or construct according to aspect 39, in which    said one or more other groups, residues, moieties or binding units    that provide the compound or construct with increased half-life is    chosen from the group consisting of human serum albumin or fragments    thereof.-   Aspect 43, Compound or construct according to aspect 39, in which    said one or more other groups, residues, moieties or binding units    that provides the compound or construct with increased half-life are    chosen from the group consisting of binding units that can bind to    serum albumin (such as human serum albumin) or a serum    immunoglobulin (such as IgG).-   Aspect 44. Compound or construct according to aspect 39, in which    said one or more other groups, residues, moieties or binding units    that provides the compound or construct with increased half-life are    chosen from the group consisting of domain antibodies, amino acid    sequences that are suitable for use as a domain antibody, single    domain antibodies, amino acid sequences that are suitable for use as    a single domain antibody, “dAb”'s, amino acid sequences that are    suitable for use as a dAb, or Nanobodies that can bind to serum    albumin (such as human serum albumin) or a serum immunoglobulin    (such as IgG).-   Aspect 45. Compound or construct according to aspect 39, in which    said one or more other binding units that provides the compound or    construct with increased half-life are chosen from SEQ ID NO's:    97-99.-   Aspect 46. Compound or construct according to any of aspects 35 to    45, selected from the following polypeptide sequences:    -   a) SEQ ID NO's 70-72;    -   b) a polypeptide sequence that has no more than 2, preferably no        more than 1 amino acid difference in one, two or all of its CDRs        of the invention with one of SEQ ID NO's: 70-72, provided that        the polypeptide sequence with no more than 2, preferably no more        than 1 amino acid difference in one, two or all of its CDRs of        the invention binds IL-6R with about the same or a higher        affinity compared to the binding by the one of SEQ ID NO's:        70-72, said affinity as measured by surface plasmon resonance;    -   c) a polypeptide sequence that has no more than 2, preferably no        more than 1 amino acid difference with one of SEQ ID NO's:        70-72, provided that the amino acid sequence with no more than        2, preferably no more than 1 amino acid difference with one of        SEQ ID NO's: 70-72 binds IL-6R with about the same or a higher        affinity compared to the binding by the one of SEQ ID NO's:        70-72, said affinity as measured by surface plasmon resonance.-   Aspect 47. Compound or construct according to any of aspects 35 to    46, selected from the following polypeptide sequences:    -   a) SEQ ID NO's 70-71;    -   b) a polypeptide sequence that has no more than 2, preferably no        more than 1 amino acid difference in one, two or all of its CDRs        of the invention with one of SEQ ID NO's: 70-71, provided that        the polypeptide sequence with no more than 2, preferably no more        than 1 amino acid difference in one, two or all of its CDRs of        the invention binds IL-6R with about the same or a higher        affinity compared to the binding by the one of SEQ ID NO's:        70-71, said affinity as measured by surface plasmon resonance;    -   c) a polypeptide sequence that has no more than 2, preferably no        more than 1 amino acid difference with one of SEQ ID NO's:        70-71, provided that the amino acid sequence with no more than        2, preferably no more than 1 amino acid difference with one of        SEQ ID NO's: 70-71 binds IL-6R with about the same or a higher        affinity compared to the binding by the one of SEQ ID NO's:        70-71, said affinity as measured by surface plasmon resonance.-   Aspect 48. Compound or construct according to any of aspects 35 to    47, that has or essentially consists of the amino acid sequence of    SEQ ID NO: 70.-   Aspect 49. Compound or construct according to any of aspects 35 to    47, that has or essentially consists of the amino acid sequence of    SEQ ID NO: 71.-   Aspect 50. Compound or construct according to any of aspects 35 to    49, that specifically binds to hIL-6R with a dissociation constant    (K_(D)) of 1 nM to 1 pM moles/litre or less, preferably 500 pM to 1    pM moles/litre or less, more preferably 100 pM to 1 pM moles/litre    or less, or even more preferably about 50 pM to 1 pM or less.-   Aspect 51. Compound or construct according to any of aspects 35 to    50, that specifically binds to cyno IL-6R with a dissociation    constant (K_(D)) of 1 nM to 1 pM or less, preferably 500 pM to 1 pM    or less, more preferably 100 pM to 1 pM or less, or even more    preferably about 50 pM to 1 pM or less.-   Aspect 52. Compound or construct according to any of aspects 35 to    51, that specifically binds to hIL-6R with a k_(on)-rate of between    10⁴ M⁻¹s⁻¹ to about 10⁷ M⁻¹s⁻¹, preferably between 10⁵ M⁻¹s⁻¹ and    10⁷ M⁻¹ s⁻¹, more preferably about 10⁶ M⁴s⁻¹ or more.-   Aspect 53. Compound or construct according to any of aspects 35 to    52, that specifically binds to cyno IL-6R with a k_(on)-rate of    between 10⁴ M⁻¹s⁻¹ to about 10⁷ M⁻¹s⁻¹, preferably between 10⁵    M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more preferably about 10⁶ M⁻¹s⁻¹ or more.-   Aspect 54. Compound or construct according to any of aspects 35 to    53, that specifically binds to hIL-6R with a k_(off) rate between    10⁻³ s⁻¹ (t_(1/2)=0.69 s) and 10⁻⁶ s⁻¹ (providing a near    irreversible complex with a t_(1/2) of multiple days), preferably    between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹, more preferably between 10⁻⁵ s⁻¹ and    10⁻⁶ s⁻¹, such as about 10⁻⁵ s⁻¹ or lower.-   Aspect 55. Compound or construct according to any of aspects 35 to    54, that specifically binds to cyno IL-6R with a k_(off) rate    between 10⁻³ s⁻¹ (t_(1/2)=0.69 s) and 10⁻⁶ s⁻¹ (providing a near    irreversible complex with a t_(1/2) of multiple days), preferably    between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹, more preferably between 10⁻⁵ s⁻¹ and    10⁻⁶ s⁻¹, such as about 10⁻⁵ s⁻¹ or lower.-   Aspect 56. Compound or construct according to any of aspects 35 to    55, that, in the TF-1 assay, has IC50 values (at 100 IU/mL IL-6)    between 10 nM and 50 pM, preferably between 5 nM and 50 pM, more    preferably between 1 nM and 50 pM or less, such as about 750 or 500    pM or less.-   Aspect 57. Compound or construct according to any of aspects 35 to    56, that, in the TF-1 assay, has IC50 (at 5000 IU/mL IL-6) between    50 nM and 1 nM, preferably between 25 nM and 1 nM, more preferably    between 10 nM and 1 nM or less, such as about 8 nM or less.-   Aspect 58. Compound or construct according to any of aspects 35 to    57, that, in the TF-1 assay, has IC50 values that are at least the    same and preferably better, at least two times, preferably three    times, more preferably four times, even more preferably 5 times, 7    times or more than 7 times better compared to the IC50 value    obtained for the reference IgG as defined by HQ ID NO's: 1 and 2 or    the reference Fab as defined by SEQ ID NO's: 3 and 4-   Aspect 59. Compound or construct according to any of aspects 35 to    58, that, in the TF-1 assay, has IC50 values that are at least the    same and preferably better, at least two times, preferably three    times, more preferably four times, even more preferably 5 times, 7    times or more than 7 times better compared to the IC50 value    obtained for Tocilizumab (MRA).-   Aspect 60. Compound or construct according to any of aspects 35 to    59, that has, in a plasma potency assay at EC50 values of IL-6, IC50    values between 500 pM and 50 pM, preferably between 250 pM and 50    pM, more preferably between 200 pM and 50 pM or less, such as 150 pM    or less,-   Aspect 61. Compound or construct according to any of aspects 35 to    60, that has, in a plasma potency assay at EC95 values of IL-6, IC50    values between 1000 pM and 100 pM, preferably between 750 pM and 100    pM, more preferably between 500 pM and 100 pM or less, such as 400    pM or less.-   Aspect 62. Compound or construct according to any of aspects 35 to    61, that has, in a plasma potency assay of aspects 60 or 61, IC50    values that are at least the same and preferably better, at least    two times, preferably three times, more preferably four times, even    more preferably 5 times, 7 times or more than 7 times better    compared to the IC50 value obtained for the reference IgG as defined    by SEQ ID NO's: 1 and 2 or the reference Fab as defined by SEQ ID    NQ's: 3 and 4.-   Aspect 63. Compound or construct according to any of aspects 35 to    62, that has, in a plasma potency assay of aspects 60 or 61, IC50    values that are at least the same and preferably better, at least    two times, preferably three times, more preferably four times, even    more preferably 5 times, 7 times or more than 7 times better    compared to the IC50 value obtained for Tocilizumab (MRA).-   Aspect 64. Compound or construct according to any of aspects 35 to    63, that has, for binding to membrane IL-6R on CHO cells, IC50    values between 10 nM and 100 pM, preferably between 5 nM and 100 pM,    more preferably between 2 nM and 10 pM or less, such as 2 nM or    less.-   Aspect 65. Monovalent construct, comprising or essentially    consisting of one amino acid sequence according to any of aspects 1    to 34.-   Aspect 66. Use of an amino acid sequence according to any of aspects    1 to 34 or a monovalent construct according to aspect 65, in    preparing a multivalent compound or construct according to any of    aspects 37 to 64.-   Aspect 67. Method for the preparation of a multivalent compound or    construct according to any of aspects 37 to 64, comprising the    linking of an amino acid sequence according to any of aspects 1 to    34 or a monovalent construct according to aspect 65 to one or more    groups, residues, moieties or binding units.-   Aspect 68. Method according to aspect 67, for the preparation of a    multivalent compound or construct according to any of aspects 37 to    64, comprising the linking of an amino acid sequence according to    any of aspects 1 to 34 or a monovalent construct according to aspect    65 to other groups, residues, moieties or binding units via one or    more linkers.-   Aspect 69. Nucleic acid or nucleotide sequence, that encodes an    amino acid sequence according to any of aspects 1 to 34, a compound    or construct according to any of aspects 35 to 64 that is such that    it can be obtained by expression of a nucleic acid or nucleotide    sequence encoding the same, or a monovalent construct according to    aspect 65.-   Aspect 70. Nucleic acid or nucleotide sequence according to aspect    69, that is in the form of a genetic construct.-   Aspect 71. Host or host cell that expresses, or that under suitable    circumstances is capable of expressing, an amino acid sequence    according to any of aspects 1 to 34, a compound or construct    according to any of aspects 35 to 64 that is such that it can be    obtained by expression of a nucleic acid or nucleotide sequence    encoding the same, or a monovalent construct according to aspect 65;    and/or that comprises a nucleic acid or nucleotide sequence    according to aspect 69, or a genetic construct according to aspect    70.-   Aspect 72. Method for producing an amino acid sequence according to    any of aspects 1 to 34, a compound or construct according to any of    aspects 35 to 64 that is such that it can be obtained by expression    of a nucleic acid or nucleotide sequence encoding the same, or a    monovalent construct according to aspects 65, said method at least    comprising the steps of:    -   a) expressing, in a suitable host cell or host organism or in        another suitable expression system, a nucleic acid or nucleotide        sequence according to aspect 69, or a genetic construct        according to aspect 70;    -   optionally followed by:    -   b) isolating and/or purifying the amino acid sequence according        to any of aspects 1 to 34, the compound or construct according        to any of aspects 35 to 64 that is such that it can be obtained        by expression of a nucleic acid or nucleotide sequence encoding        the same, or the monovalent construct according to aspect 65        thus obtained.-   Aspect 73. Method for producing an amino acid sequence according to    any of aspects 1 to 34, a compound or construct according to any of    aspects 35 to 64 that is such that it can be obtained by expression    of a nucleic acid or nucleotide sequence encoding the same, or a    monovalent construct according to aspect 65, said method at least    comprising the steps of:    -   a) cultivating and/or maintaining a host or host cell according        to aspect 71 under conditions that are such that said host or        host cell expresses and/or produces at least one amino acid        sequence according to any of aspects 1 to 34, compound or        construct according to any of aspects 35 to 64 that is such that        it can be obtained by expression of a nucleic acid or nucleotide        sequence encoding the same, or a monovalent construct according        to aspect 65,    -   optionally followed by:    -   b) isolating and/or purifying the amino acid sequence according        to any of aspects 1 to 34, the compound or construct according        to any of aspects 35 to 64 that is such that it can be obtained        by expression of a nucleic acid or nucleotide sequence encoding        the same, or the monovalent construct according to aspect 65        thus obtained.-   Aspect 74. Composition, comprising at least one amino acid sequence    according to any of aspects 1 to 34, compound or construct according    to any of aspects 35 to 64, monovalent construct according to aspect    65, or nucleic acid or nucleotide sequence according to aspects 69    or 70.-   Aspect 75. Composition according to aspect 74, which is a    pharmaceutical composition.-   Aspect 76. Composition according to aspect 74, which is a    pharmaceutical composition, that further comprises at least one    pharmaceutically acceptable carrier, diluent or excipient and/or    adjuvant, and that optionally comprises one or more further    pharmaceutically active polypeptides and/or compounds.-   Aspect 77. Method for the prevention and/or treatment of at least    one of the diseases and disorders associated with IL-6, with IL-6R,    with the IL-6/IL-6R complex and/or with the signalling pathways    and/or the biological functions and responses in which IL-6, IL-6R    and/or the IL-6/IL-6R complex are involved, said method comprising    administering, to a subject in need thereof, a pharmaceutically    active amount of at least one amino acid sequence according to any    of aspects 1 to 34, a compound or construct according to any of    aspects 35 to 64, or a monovalent construct according to aspect 65,    or composition according to any of aspects 75 to 76.-   Aspect 78. Method according to aspect 77, wherein said diseases and    disorders associated with IL-6R and/or with the IL-6/IL-6R complex    and/or with the signaling pathways and/or the biological functions    and responses in which IL-6 and/or the IL-6/IL-6R complex are    involved are chosen from the group consisting of sepsis, various    forms of cancer, bone resorption, osteoporosis, cachexia, psoriasis,    mesangial proliferative glomerulonephritis, Kaposi's sarcoma,    AIDS-related lymphoma, and inflammatory diseases.-   Aspect 79. Method according to aspect 78, wherein said various forms    of cancer are chosen from the group consisting of multiple myeloma    disease (MM), renal cell carcinoma (RCC), plasma cell leukaemia,    lymphoma, B-lymphoproliferative disorder (BLPD), and prostate    cancer.-   Aspect 80. Method according to aspect 78, wherein said inflammatory    diseases are chosen from the group consisting of rheumatoid    arthritis, systemic onset juvenile idiopathic arthritis,    hypergammaglobulinemia, Crohn's disease, ulcerative colitis,    systemic lupus erythematosus (SLE), multiple sclerosis, Castleman's    disease, IgM gammopathy, cardiac myxoma, asthma, allergic asthma and    autoimmune insulin-dependent diabetes mellitus.-   Aspect 81. Method for the prevention and/or treatment of at least    one disease or disorder that can be prevented and/or treated by    administering, to a subject in need thereof, an amino acid sequence    according to any of aspects 1 to 34, a compound or construct    according to any of aspects 35 to 64, or a monovalent construct    according to aspect 65, said method comprising administering, to a    subject in need thereof, a pharmaceutically active amount of at    least one amino acid sequence according to any of aspects 1 to 34,    compound or construct according to any of aspects 35 to 64, or    monovalent construct according to aspect 65, or composition    according to any of aspects 75 to 76.-   Aspect 82. Use of an amino acid sequence according to any of aspects    1 to 34, compound or construct according to any of aspects 35 to 64,    or monovalent construct according to aspect 65, in the preparation    of a pharmaceutical composition for prevention and/or treatment of    at least one of the diseases and disorders associated with IL-6,    with IL-6R, with the IL-6/IL-6R complex and/or with the signalling    pathways and/or the biological functions and responses in which    IL-6, IL-6R and/or the IL-6/IL-6R complex are involved; and/or for    use in one or more of the methods according to aspects 77 to 81.-   Aspect 83. An amino acid sequence according to any of aspects 1 to    34, a compound or construct according to any of aspects 35 to 64, or    monovalent construct according to aspect 65 for use in the    prevention and/or treatment of at least one of the diseases and    disorders associated with IL-6, with IL-6R, with the IL-6/IL-613    complex and/or with the signalling pathways and/or the biological    functions and responses in which IL-6, IL-6R and/or the IL-6/IL-6R    complex are involved; and/or for use in one or more of the methods    according to aspects 77 to 81.-   Aspect 84. Derivative of an amino acid sequence according to any of    aspects 1 to 34, a compound or construct according to any of aspects    35 to 64, or monovalent construct according to aspect 65.-   Aspect 85. Derivative according to aspect 84, that can specifically    bind to IL-6R.-   Aspect 86. Derivative according to any of aspects 84 to 85, that has    a serum half-life that is at least 1.5 times, preferably at least 2    times, such as at least 5 times, for example at least 10 times or    more than 20 times, greater than the half-life of the amino acid    sequence according to any of aspects 1 to 34 per se, compound or    construct according to any of aspects 35 to 64 per se, or monovalent    construct according to aspect 65 per se.-   Aspect 87. Derivative according to any of aspects 84 to 86, that has    a serum half-life that is increased with more than 1 hours,    preferably more than 2 hours, more preferably more than 6 hours,    such as more than 12 hours, or even more than 24, 48 or 72 hours,    compared to the corresponding amino acid sequence according to any    of aspects 1 to 34 per se, a compound or construct according to any    of aspects 35 to 64 per se, or monovalent construct according to    aspect 65 per se.-   Aspect 88. Derivative according to any of aspects 84 to 87, that has    a serum half-life in human of at least about 12 hours, preferably at    least 24 hours, more preferably at least 48 hours, even more    preferably at least 72 hours or more; for example, at least 5 days    (such as about 5 to 10 days), preferably at least 9 days (such as    about 9 to 14 days), more preferably at least about 10 days (such as    about 10 to 15 days), or at least about n days (such as about 11 to    16 days), more preferably at least about 12 days (such as about 12    to 18 days or more), or more than 14 days (such as about 14 to 19    days).-   Aspect 89. Derivative according to any of aspects 84 to 88, that is    a pegylated derivative.-   Aspect 90. Composition, comprising at least one derivative according    to any of aspects 84 to 89.

EXAMPLES I. Isolation of IL-6R Binding Nanobodies Example 1 Materials

The materials used for the isolation of EL-6R binding Nanobodies aregiven in Table C-1. Two representative anti-human IL-6R immunoglobulinsdescribed in EP 0628639 (a Fab fragment and a full-sized IgG) weregenerated and used as reference compounds. The Fab fragment andfull-sized IgG were constructed based on the L-chain called “RV_(L)a”(see EP 0628639 B1, Table 2, version (a)) and the H-chain called“RV_(H)f” (see EP 0628639131, Table 3, version (f)). These particularL-chain and H-chain were chosen for the purposes of constructing thereference compounds because, according to EP 0628639 B1 (see for exampleparagraph [0074]), a reshaped human antibody comprising said L-chain andsaid H-chain exhibited an ability to bind to human IL-6R at the samelevel as PM1, a mouse monoclonal antibody against human IL-6R (see againEP 0628639 B1, paragraph and the further references cited therein).

The full-length reference IgG consisted of the amino acid sequences ofSEQ ID NO: 1 (heavy chain) and SEQ ID NO: 2 (light chain). The Fabfragment consisted of the amino acid sequences of SEQ ID NO: 3 (heavychain regions V_(L)b and V_(H)f fused to the CH1 region of human IgG1)and SEQ ID NO: 4 (reshaped human PM-1 variable light chain fused tohuman Ckappa).

Encoding DNA fragments were generated by assembly PCR using partiallyoverlapping oligonucleotides. PCR products were cloned into a single,bi-cistronic vector which enables expression of functional,disulphide-linked Fab fragments in the periplasm of E. coli. Full-lengthIgG was produced in CHO cells transfected with 2 expression vectorscontaining the genes for the light and heavy chains. The gene encodingthe heavy chain was created by fusing V_(H)f to the constant region ofhuman IgG1. The light chain was as described in EP 0628639.

Example 2 Immunizations

Two llamas (81 and 82) were immunized with human IL-6R (Peprotech)according to the immunization schedule described in Table C-2.

After completion of the immunization schedule the immune response ineach animal was analyzed by ELISA. To this end, biotinylated IL-6R (2μg/ml) was captured in a neutravidin coated microtiter plate. Serialdilutions of serum samples collected at days 0, 28, 39 and 43 were added(starting dilution: 1/500) and bound llama IgG was detected by additionof goat anti-llama IgG HRP labeled. TMB was used as a substrate. Resultsare shown in FIG. 1.

immune responses were also analyzed by FACS: serial dilutions (startingdilution: 1/100) of serum samples collected at days 0, 28 and 43 wereincubated with U266 cells (human myeloma). Bound llama IgG was detectedby goat anti-llama IgG FITC labeled. Results are shown in FIG. 2.

Together these data show that both animals generated a good immuneresponse against IL-6R and that at least a fraction of llama IgGrecognize IL-6R on the surface of U266 cells.

Example 3 Library Construction

RNA extracted from PBLs and lymph node was used as starting material forRT-PCR to amplify Nanobody encoding gene fragments. These fragments werecloned into an expression vector derived from pUC119 which contained theLacZ promoter, a coliphage pill protein coding sequence, a resistancegene for ampicillin or carbenicillin, a multicloning site and the gen3leader sequence. In frame with the Nanobody coding sequence, the vectorcoded for a C-terminal c-myc tag and a (His)6 tag. Phage was preparedaccording to a standard protocol and stored after filter sterilizationat 4° C. for further use. The characteristics of the constructedlibraries are shown in Table C-3.

Example 4 Selections

Selections were carried out with the above libraries using variousconditions as summarized in Table C-4.

Only a single round of selection was performed for all conditions. Eachselection output was analyzed for enrichment factor (# phage present ineluate relative to control), diversity (HinfI profiling) and percentageof IL-6R positive clones (ELISA). Based on these parameters the bestselections were chosen for further analysis. To this end, the outputfrom each selection was recloned as a pool into an expression vectorderived from pUC119 which contained the LacZ promoter, a resistance genefor ampicillin or carbenicillin, a multicloning site and the gen3 leadersequence. In frame with the Nanobody coding sequence, the vector codedfor a C-terminal c-myc tag and a (His)6 tag. Colonies were picked andgrown in 96 deep well plates (1 ml volume) and induced by adding IPTGfor Nanobody expression. Periplasmic extracts were prepared according tothe standard protocol.

Example 5 Screening

The periplasmic extracts were analyzed first for their ability toinhibit the IL-6-IL-6R interaction. To this end, 2 independentAlphascreen assays were set up which are depicted schematically in FIG.3. In assay 1, the periplasmic extracts were incubated with soluble IL-6receptor (1 nM), biotinylated IL-6 (3 nM), streptavidin coated donorbeads and MAb BN-12 coated acceptor beads (20 μg/ml). Nanobodiespositive in this assay could either inhibit the IL-6/IL-6R interactionor IL-6R-MAb BN-12 interaction. To discriminate between these 2possibilities a second assay was set up (Assay 2). In this assay theperiplasmic extract were incubated with bio-IL-6R (0.3 nM), streptavidincoated donor beads and MAb BN-12 coated acceptor beads (10 μg/ml).Nanobodies positive in assay 1 but negative in assay 2 were consideredas IL-6-IL-6R inhibitors.

Periplasmic extracts were diluted 25-fold in both assays whichcorresponds roughly to a final concentration of 40 nM. A statisticaloverview of the screening effort is shown in Table C-5 below. Nanobodiesshowing the strongest inhibition were selected for off-rate analysis onBiacore and DNA sequencing. FIG. 4 shows protein sequences of inhibitoryNanobodies that were selected for further analysis in cell based assays.Table C-6 shows k_(off)-values of these inhibitory Nanobodies.

Example 6 Nanobody Expression and Purification

Selected Nanobodies were expressed in E. coli as c-myc, (His)6-taggedproteins in a culture volume of 50 or 250 ml. Expression was induced byaddition of 1 mM IPTG and allowed to continue for 4 h at 37° C. Afterspinning the cell cultures, periplasmic extracts were prepared byfreeze-thawing the pellets. These extracts were used as startingmaterial for immobilized metal affinity chromatography (IMAC).Nanobodies were eluted from the column with 150 mM imidazole andsubsequently dialyzed against PBS. Total yield and yield per litre ofcell culture are listed in Table C-7. SDS-PAGE of purified Nanobodies(except for PMP28E11) is shown in FIG. 5.

Example 7 Protein Based Competition Assay

The 14 purified Nanobodies were tested in Alphascreen for inhibition ofthe IL-6/IL-6R interaction. Serial dilutions of purified proteins(concentration range: 500 nM-10 pM) were added to IL-6R (0.3 nM) andincubated for 15 min. Subsequently 3 nM bio-IL-6 and BN-12-coatedacceptor beads were added and this mixture was incubated for 1 hour.Finally streptavidin donor beads were added and after 1 hour incubatorthe plate was read on the Envision microplate reader. BR-6 and the Fabfragment described in Example 1 were included as reference. Results areshown in FIG. 6.

Dose-response curves were observed for all 14 Nanobodies withIC₅₀-values ranging from 48 pM to 1.7 nM (Table C-8). The most potentNanobodies in this assay were PMP32C9 and PMP35H4. For PMP33A3 onlypartial (˜50%) inhibition of IL-6/IL-6R interaction could be achieved.

Example 8 Affinity Determination of the Nanobodies Obtained

Affinity constants (Kd) of individual Nanobodies and the reference Fabfragment described in Example 1 were determined by surface plasmonresonance (SPR) on a Biacore 3000 instrument. In brief, IL-6R wasamine-coupled to a CM5 sensor chip at a density of 800-1000 RU.Nanobodies were injected at 5 different concentrations between 1 and 50nM. Flow rate was 45 μl/min in all experiments. Association anddissociation phase were 3 and 10 min, respectively. The chip wasregenerated using Glycine/HCl pH 1.5. Binding curves at differentconcentrations of Nanobody were used to calculate the kinetic parametersk_(on), k_(off) and K_(d) (Table C-9).

Example 9 Cell Based Potency of the Nanobodies in XG1 Assay

All purified Nanobodies were tested in the XG1 assay. XG1 is anIL-6-dependent human myeloma cell line. Half-maximal proliferation isachieved at ˜20 pg/ml of IL-6. Assays were essentially performed asdescribed by Zhang et al. (1994, Blood 83: 3654-3663). The reference Fabfragment as described in Example 1 was included as a reference. IC50values ranged from 90 pM to 50 nM as listed in Table C-10. A smallsubset of Nanobodies was also tested in this assay in the presence of 1mg/ml HSA.

Example 10 Cell Based Potency of the Nanobodies in TF1 Assay

Nanobodies were also tested for their ability to inhibit IL-6-dependentproliferation of TF-1 cells (ECACC no. 93022307; 1989, J. Cell Physiol.,140: 323; 1993, Exp, Cell Res., 208: 35) by blocking of IL-6 binding toIL-6R on the cell-surface. To this end, serial dilutions of Nanobodywere pre-incubated with a fixed amount of TF-1 cells for 2 hours at 37°C. Subsequently IL-6 was added to a final concentration of 2 ng/ml.IL-6-dependent cell proliferation was allowed to continue for 72 hoursand was measured by the incorporation of tritium labeled thymidine. IC50values are listed in Table C-11.

Example 11 Competition with the Reference Fab for Binding to IL-6R

All 14 Nanobodies were analyzed for their ability to inhibit the bindingof the reference Fab as described in Example 1 to IL-6R in anAlphascreen based assay. In this assay, 100 nM of purified Nanobody wasincubated with 0.4 nM of biotinylated IL-6R. Reference-Fab coatedacceptor beads and streptavidin coated donor beads were added and theconcentration of Reference-Fab/IL-6R complex was measured. Valuesobtained in the presence of Nanobody were compared to a control where noNanobody was added and the ratios between the 2 values, expressed as %,are listed in Table C-12. All Nanobodies, except IL6R03, show no or onlypartial inhibition of the reference Fab binding to IL-6R, suggestingthat their epitopes do not or only partially overlap with the epitope ofthe reference Fab.

Example 12 Binding of the Nanobodies to U266 Cells

Binding of the Nanobodies to membrane-bound IL-6R expressed on U266cells was analyzed in FACS. The analysis was done for purifiedNanobodies from selected clones (IL6R04, IL6R09, IL6R11, IL6R13 andIL6R14). Results are shown in FIG. 7. All Nanobodies were able to bindto cell-surface expressed human IL-6R.

Example 13 Binding of the Nanobodies to Plasma-Derived Human IL-6R

Soluble IL-6R is present in human plasma at a concentration of 80-400ng/ml. To determine whether Nanobodies IL6R03, IL6R04 and IL6R13 areable to bind plasma derived IL-6R, the effect of human plasma onNanobody binding to U266 cells was evaluated. Human plasma inhibitedbinding to U266 cells, indicating that all 3 Nanobodies are able to bindto plasma-derived human IL-6R (see FIG. 8).

Example 14 Cross-Reactivity of the Nanobodies to Mouse IL-6R

Cross-reactivity of the Nanobodies to mouse IL-6R was analyzed in ELISA.To this end, 500 nM of the Nanobodies was applied to a microtiter platecoated with 1 μg/ml mouse and human IL-6R. Detection was performed withanti-myc and anti-mouse-HRP as first and second antibody respectively.Optical densities are shown in FIG. 10. No binding to mouse IL-6R wasobserved for any of the Nanobodies tested.

Example 15 Summary of the Isolation of IL-6R Binding Nanobodies

Immunization of 2 llamas with recombinant IL-6R resulted in a panel of14 unique Nanobodies which were able to block the interaction betweenIL-6 and IL-6R. This panel was analyzed in detail and based on all theexperimental data Nanobodies IL6R03, IL6R04 and IL6R13 were selected forfurther development. The most important Nanobody characteristics aresummarized in Table C-13.

II. Formatting of the Anti-IL-6R Nanobodies Example 16 Preparation ofMultivalent Constructs

The anti-IL-6R Nanobodies described in the previous paragraphs were alsoexpressed as bispecific constructs consisting of a C-terminal anti-SANanobody (ALB1), a 9 amino acid Gly/Ser linker and an N-terminalanti-IL-6R Nanobody. In addition, 4 trivalent, bispecific Nanobodieswere constructed consisting of a C-terminal and N-terminal anti-IL-6RNanobody, an anti-SA Nanobody (ALB1) in the middle, all connected via 9amino acid Gly/Ser linkers. The IDs of these Nanobodies are listed inTable C-14.

Example 17 Expression of Bispecific Anti-IL-6R Nanobodies

Bispecific Nanobody constructs were expressed in E. coli as c-myc,(His)6-tagged proteins and subsequently purified from the culture mediumby immobilized metal affinity chromatography (IMAC) and size exclusionchromotography (SEC). Total yield and yield per litre of cell cultureare listed in the Table C-15. SDS-PAGE of purified Nanobodies is shownin FIG. 11.

Example 18 Protein Based Competition Assay

The purified bispecific Nanobodies were tested in Alphascreen forinhibition of the IL-6/IL-6R interaction. Serial dilutions of purifiedproteins (concentration range: 250 nM-5 pM) were added to IL-6R (0.3 nM)and incubated for 15 min. Subsequently 3 nM bio-IL-6 and BN-12-coatedacceptor beads were added and this mixture was incubated for 1 hour.Finally streptavidin donor beads were added and after 1 hour incubatorthe plate was read on the Envision microplate reader. BR-6 and the Fabfragment described in Example 1 were included as reference. Results areshown in FIG. 12.

Dose-response curves were observed for all Nanobodies with IC₅₀-valuesranging from 123 pM to 1.67 nM (Table C-16).

Example 19 Cell Based Potency of the Nanobodies in XG1 Assay

Bispecific Nanobodies were tested in the XG1 proliferation assay. IC50values ranged from 60 pM to 65 nM. Nanobodies were also analyzed in thisassay in the presence of 1 mg/mL human serum albumin. IC50 values rangefrom 190 pM to 90 nM for bispecific Nanobodies. The reference IgG asdescribed in Example 1 was included as reference. IC50 values are listedin Table C-17.

A loss in potency was observed when formatted Nanobodies were tested inthe XG1 assay in the presence of albumin. Potencies of formattedNanobodies IL6R24, IL6R44 and IL6R49 were superior to or in the samerange as the reference IgG in the presence of serum albumin.

Example 20 Determination of Affinity for IL-6R

Binding of bispecific Nanobodies to IL-6R was analyzed by surfaceplasmon resonance. Kinetic parameters were determined and are listed inTable C-18. No significant loss in affinity for IL-6R was observed forthe Nanobodies in bivalent format (IL6R23, IL6R24, IL6R33).

Example 21 Determination of Affinity for Serum Albumin

Binding of formatted Nanobodies to serum albumin was analyzed by surfaceplasmon resonance. Affinity constants (Kd) were determined and arelisted in Table C-19. The albumin binding Nanobody Alb-1 (SEQ ID NO: 97)was included for comparison. The affinities were in the range ofpreviously formatted Nanobodies containing the same anti-serum albuminbuilding block, however in general a lower affinity was observed. Thiswas particularly the case for mouse serum albumin affinity.

Example 22 Binding of the Formatted Nanobodies to U266 Cells

Binding of formatted Nanobodies from selected clones (IL6R23, IL6R24,IL6R29, IL6R33, IL6R44 and IL6R53) to U266 cells was analyzed by FAGS.Results are shown in FIGS. 13 and 14. Bivalent Nanobodies IL6R23,IL6R24, IL6R29 and IL6R33 show reduced binding as compared to themonovalent building blocks, whereas similar binding was observed fortrivalent Nanobodies IL6R44 and IL6R53.

III. Sequence Optimization of Anti-IL-6R Nanobodies Example 23 SequenceOptimization Strategy

Protein sequences of Nanobodies IL6R03, IL6R04 and IL6R13 were eachaligned with the 5 closest human germ lines sharing the highest degreeof homology (FIG. 15). Amino acid differences in the framework regionsrelative to the human germline consensus sequence are color coded. Aminoacid differences highlighted in light gray were selected for conversioninto the human counterpart whereas amino acids highlighted in dark graywere left untouched. Initially a panel of 4 sequence optimized variantswas generated for each of the 3 Nanobodies (Stage 1). These variantswere analyzed for a number of parameters and the results obtained wereused to design a second set of Nanobodies (Stage 2). Protein sequencesof all sequence optimized variants are shown in FIG. 16.

Example 24 Sequence Optimization Stage 1

In stage 1 of the sequence optimization process the following 12variants were created and analyzed:

IL6R03: IL6R61, IL6R62, IL6R63 and IL6R64 IL6R04: IL6R71, IL6R72, IL6R73and IL6R74 IL6R13: IL6R81, IL6R82, IL6R83 and IL6R84

Amino acid sequences of these different variants are shown in FIG. 16.

Evaluation of Sequence Optimized Nanobodies in the IL-6/IL-6RCompetition Assay

Sequence optimized clones of IL6R03, IL6R04 and IL6R13 were tested inAlphascreen for inhibition of the IL-6/IL-6R interaction. Serialdilutions of purified Nanobodies were added to IL-6R (03 nM) andincubated for 15 min. Subsequently 3 nM bio-IL-6 and BN-12-coatedacceptor beads were added and this mixture was incubated for 1 hour.Finally streptavidin donor beads were added and after 1 hour incubationthe plate was read on the Envision microplate reader. Parental cloneswere included as reference. Results are shown in FIG. 17, FIG. 18 andFIG. 19.

The sequence optimized variants of IL6R03 (IL6R61, 62 and 64) have IC50values all within 2-fold of the IC50 value of IL6R03, whereas IL6R63displays a ˜4-fold lower IC50 value.

For the sequence optimized variants of IL6R04, no significantdifferences were observed in IC50 values between 116R04 and the 4sequence optimized variants.

For the sequence optimized variants of IL6R13, the IC50 values of IL6R81and IL6R83 were almost identical to that of IL6R13 while the 2 variantscarrying the RAT→KGL mutation in framework 2 (IL6R82 and IL6R84) had adrop in potency.

Affinity Determination

The sequence optimized variants of IL6R03, IL6R04 and IL6R13 were alsoanalyzed on Biacore for binding to IL-6R. Kinetic parameters are listedin Table C-20.

For the sequence optimized variants of IL6R03, K_(d) values of IL6R61,62 and 63 were all within 2-fold of the K_(d) value of IL6R03. The K_(d)of IL6R64 was not determined.

For the sequence optimized variants of IL6R04, no significantdifferences were observed in K_(d) values between IL6R04 and the 4sequence optimized variants.

For the sequence optimized variants of IL6R13, K_(d) values of IL6R81and IL6R83 are very similar to the K_(d) of IL6R13 while the 2 variantscarrying the RAT→KGL mutation in framework 2 (IL6R82 and IL6R84)displayed a serious drop in affinity.

Overall, these observations are in perfect agreement with the resultsobtained in the Alphascreen based competition assay.

Example 25 Sequence Optimization Stage 2

Based on the affinity and potency data of stage 1 it was decided togenerate the following set of variants:

-   -   For IL6R03 all mutations present in variants IL6R61, 62, 63 and        64 were combined to yield ILR65.    -   For 16R04 all mutations present in variants IL6R71, 72, 73 and        74 were combined to yield ILR7.5.    -   For IL6R13 a set of 6 new variants (IL6R85-90) was created        carrying different (combinations of) mutations in the RAT        sequence in FR2. These mutations were introduced in the IL6R83        background as this sequence optimized variant was        indistinguishable from IL6R13 in both affinity and potency.

Off-Rate Analysis of Sequence Optimized IL6R13 Variants

Sequence optimized variants IL6R85-90 were analyzed as periplasmicextracts on Biacore. Dissociation curves were used to calculate k_(af)values (Table C-21). Off-rates of Nanobodies IL6R87-89 were similar tothat of IL6R13 while Nanobodies IL6R85, 86 and 90 had a 10-fold higheroff-rate.

Evaluation of Sequence Optimized Nanobodies in the IL-6/IL-6RCompetition Assay

Sequence optimized variants IL6R85-90 were tested as periplasmicextracts for their ability to block the IL-6/IL-6R interaction inAlphascreen. Results are shown in FIG. 20.

Nanobodies IL6R87, 88 and 89 were more potent than IL6R13 variants 85,86 and 90 in blocking the IL-6/IL-6R interaction. These results are inperfect agreement with the observations on Biacore. Comparison of theamino acid sequence of the sequence optimized IL6R13 variants revealedthat mutation T45L was responsible for the reduction in off-rate andpotency. Therefore the most human variant without the T45L mutation,i.e. IL6R88, was selected for further characterization.

This variant was expressed, purified and analyzed for inhibition of theIL-6/IL-6R interaction along with purified clones IL6R65 and IL6R75. Nosignificant differences between the different sequence optimized clones(IL6R65, IL6R75 and IL6R88) and their corresponding non-sequenceoptimized versions (IL6R03, IL6R04 and IL6R13, respectively) wasobserved (FIG. 21).

Affinity Determination

Affinity constants (K_(d)) of sequence optimized clones of IL6R65, 75and 88 for human IL-6R were determined by surface plasmon resonance on aBiacore 3000 instrument. In brief, human IL-6R was amine-coupled to aCM5 sensor chip at a density of 800-1000 RU. Remaining reactive groupswere inactivated. Nanobody binding was assessed at variousconcentrations ranging from 0.5 to 50 nM. Each sample was injected for 4min at a flow rate of 45 μl/min to allow for binding to chip-boundantigen. Next, binding buffer without Nanobody was sent over the chip atthe same flow rate to allow for dissociation of bound Nanobody. Kineticparameters of the sequence optimized variants of IL6R03, 04 and 13 aregiven in Table C-22.

No major differences in affinity constants were observed betweenparental and sequence optimized Nanobodies.

Cell-Based Potency Assay

Sequence optimized Nanobodies were analyzed in the XG-1 assay. Resultsare shown in FIG. 22. IC50 values are summarized in Table C-23. Sequenceoptimization had no significant effect on the potency to neutralizeIL-6-induced proliferation in cell-based assay.

IV. Additional Characterization of Sequence Optimized Nanobodies Example26 Affinity Determination for Cyno IL-6R

Affinity of IL6R03-IL6R65, IL6R04-IL6R75 and IL6R13-IL-6R88 for cynoIL-6R was determined by SPR on a Biacore 3000 instrument. In brief, cynoIL-6R was amine-coupled to a CM5 sensor chip at a density of 760 RU.Remaining reactive groups were inactivated. Nanobody binding wasassessed at various concentrations ranging from 1.25 to 100 nM. Eachsample was injected for 4 min at a flow rate of 45 μl/min to allow forbinding to chip-bound antigen. Next, binding buffer without Nanobody wassent over the chip at the same flow rate to allow for dissociation ofbound Nanobody. The kinetic parameters are summarized in Table C-24.

Although there was quite some difference in the affinity constants ofIL6R04 and IL6R75, from this experiment it was clear that the affinityof both molecules for cyno IL-6R was much lower than for human. Incontrast, the affinity constants of IL6R03 and IL6R65 for cyno IL-6Rwere in the same range as for human IL-6R. Strikingly, the crystalstructure of the IL-6/IL-6R/gp130 complex (Boulanger et al.) revealsthat the IL-6 binding site on IL-6R is completely conserved betweenhuman and cyno, suggesting that IL6R04 is binding to a differentepitope.

Example 27 Testing of the Sequence Optimized Nanobodies in PlasmaPotency Assay Using Human and Cyno Plasma

In order to assess the cross-reactivity of IL6R65 and IL6R75 withcynomolgus monkey IL-6R the plasma potency ELISA was performed usingeither human or cyno plasma as a source of sIL-6R. In this assay, adilution series of the Nanobodies was pre-incubated with plasma andhuman IL-6 (50 ng/mL). Subsequently, plasma sIL-6R was captured on aBN-12 coated plate and bound IL-6 was detected using biotinylatedpolyclonal antibodies and streptavidin-HRP.

As can be observed in FIG. 23A, both IL6R201 and IL6R65 were able tocompletely block the binding of IL-6 to human sIL-6R, but IL6R65appeared to be less potent than IL6R201, and than the reference IgG andreference Fab described in Example 1. As can be observed in FIG. 23B,IL6R65 was able to completely block the binding of IL-6 to cyno sIL-6Rwith a potency comparable to the reference Fab described in Example 1.

Example 28 Testing of the Sequence Optimized Nanobodies in PlasmaPotency Assay at High IL-6 Concentration

The plasma potency assay in human plasma was also used to test theability of the Nanobodies to block high concentrations of IL-6. IL6R04,IL6R65 and the reference Fab described in Example 1 were tested at theEC50 of IL-6 (50 ng/mL) and at the EC95 of IL-6 (885 ng/mL). The resultsare depicted in FIG. 24. Clearly, IL6R04 appeared to be the most potentcompound at the EC50 of IL-6. At the EC95 the reference Fab and IL6R65could still completely block plasma sIL-6R, albeit at higherconcentrations, indicating that these 2 molecules bind to an epitopethat overlaps with the IL-6 binding site. The IC50 of the reference Fabincreased from 0.55 nM to 8.47 nM and the IC₅₀ of IL6R6S increased from2.61 nM to 66.25 nM.

Example 29 Biacore Competition Studies

Biacore experiments were carried out to investigate whether IL-6 andIL6R65 were able to bind to IL-6R simultaneously. The reference Fab(FIG. 25A), 16R201/75 (FIG. 25B) or IL6R65 (FIG. 25C) were captured onIL-6R, after which IL-6 was allowed to bind to the complex. With thereference Fab and IL6R65 almost no binding of IL-6 could be observed.When IL-6 was captured on IL-6R and the Nanobodies were injected, allthree Nanobodies seemed to be able to bind to the complex. However, inthe case of the reference Fab and IL-6R65 this was probably because IL-6was displaced due to the lower affinity of IL-6 for IL-6R.

Finally, IL-6R was allowed to bind to an IL-6 coated chip in thepresence or absence of Nanobodies (FIG. 25D). This confirmed the earlierresults, namely IL-6R could not be captured in the presence of thereference Fab or IL6R65.

in conclusion, the Biacore epitope mapping experiments showed that boththe reference Fab and IL6R65 target the same epitope as IL-6. WhileIL6R65 and the reference Fab were able to block the binding of IL-6 toIL-6R completely, IL6R201 was unable to prevent binding of IL-6 to IL-6Rwhen this Nanobody was bound to the receptor.

V. Affinity Maturation of IL6R65 Example 30 Diversification Strategy forAffinity Maturation

CDR regions of the Nanobody IL6R65 were randomized using the 2 followingstrategies:

-   1. Each CDR residue was replaced by a set of residues with similar    side-chain chemistries:

K

R

A

S

T

I

L

V

F

Y

N

D

Q

E

G→A

M→L

H,C,W,P were kept constant

-   2. Each CDR residue was replaced by a panel of amino acids which    naturally occur on that position. Only the most frequently occurring    amino acids on each position were introduced in order to limit the    diversity per position. This approach was only used for    randomization of CDR1 and CDR2.

Concurrent randomization of CDR1 and 2 was performed using the 2strategies described above and CDR3 was randomized separately followingstrategy 1 resulting in a total number of 3 libraries. All 3 librarieswere made in-house by PCR overlap extension using degenerate oligos.Theoretical diversity for each of the libraries was approximately1×10e6. Fragments encoding the Nanobody variants were cloned into thephage display vector. The actual size of all 3 libraries was around1×10e8 (100× coverage of theoretical diversity). One round of selectionwas performed using different concentration of biotinylated IL-6R (0, 1,10 and 100 pM) in solution. No enrichment was observed for the CDR3library under these conditions while dose-dependent enrichment wasobserved for both CDR1/2 libraries. Outputs from the CDR1/2 librarieswere analyzed as periplasmic extracts in ELISA and clones with highestsignals were subsequently tested on Biacore. The top 30 clones in ELISAshowed off-rates between 2.1×10e−3 and 2.6×10e−4 s⁻¹. The 5 Nanobodieswith the slowest off-rates were sequenced, expressed and purified (FIG.26).

Example 31 Nanobody Expression and Purification

Affinity matured Nanobodies were expressed in E. coli as c-myc,(His)6-tagged proteins in a culture volume of 250 ml. Expression wasinduced by addition of 1 mM IPTG and allowed to continue for 4 h at 37°C. After spinning the cell cultures, periplasmic extracts were preparedby freeze-thawing the pellets. These extracts were used as startingmaterial for immobilized metal affinity chromatography (IMAC).Nanobodies were eluted from the column with 150 mM imidazole andsubsequently desalted on a Hiprep26/10 column.

Example 32 Affinity Determination of the Affinity Matured Variants onBiacore

Nanobody IL6R65 (sequence optimized) and the 5 affinity matured variantswere analyzed on Biacore. Binding curves at different concentrations ofpurified Nanobody were recorded and used to calculate the affinityconstants. Kd-values for these 5 clones were between 0.34 and 0.95 nMwhich corresponds to a 13-fold improvement relative to IL6R65 (parentNanobody) for the best variant (FIG. 27).

Example 33 Evaluation of the Affinity Matured Variants in Plasma PotencyAssay

All 5 affinity matured Nanobodies and the sequence optimized Nanobodywere also tested in a plasma potency assay. In this assay differentconcentrations of Nanobody were mixed with soluble IL-6R containingplasma from either human or cynomolgus monkey and a fixed concentrationof human IL-6 (2.4 or 42 nM). After 1 hour of incubation the mixture wastransferred to a Maxisorp plate coated with the anti-IL-6R MAb 13N-12(Diaclone), The amount of IL-6 bound was determined by subsequentaddition of biotinylated anti-IL-6 polyclonal antibody (R&D Systems) andstreptavidin-HRP. TMB was used as substrate. Substrate conversion wasmeasured at 450 nm (FIG. 28).

Example 34 Evaluation of the Affinity Matured Variants in TF-1 Assay

The affinity matured Nanobodies were also tested for their ability toinhibit IL-6-dependent proliferation of TF-1 cells (ECACC no. 93022307;1989, J. Cell Physiol. 140: 323; 1993, Exp. Cell Res. 208: 35) due toblocking of IL-6 binding to IL-6R on the cell-surface. To this end,serial dilutions of Nanobody were pre-incubated with a fixed amount ofTF-1 cells for 2 hours at 37 C. Subsequently IL-6 was added to a finalconcentration of 2 ng/ml. IL-6-dependent cell proliferation was allowedto continue for 72 hours and was measured by the incorporation oftritium labeled thymidine (FIG. 29).

IC50 values of affinity matured Nanobodies were up to 17-fold betterthan that of IL6R65 but all variants were still less potent than thereference-IgG.

Example 35 Biacore Competition Studies

Biacore experiments were carried out to investigate whether IL-6 is ableto bind to IL-6R simultaneously with IL6R65 and with 2 of its affinitymatured variants (7D6 and 7C4). In these experiments IL-6R was capturedon a BN-12 coated chip and saturated with Nanobody IL6R65 (FIG. 30A),7D6 (FIG. 30B) or 7C4 (FIG. 30C), Next, binding of IL-6 to theIL-6R-Nanobody complex was assessed by injection of the cytokine at aconcentration of 100 nM. In addition, also binding of the 3 Nanobodiesto IL-6R in complex with IL-6 was determined.

The results for IL6R65 (FIG. 30A) are comparable to the results observedpreviously and confirm that IL6R65 and IL-6 recognize the same epitopeon IL-6R. As expected the IL6R65 affinity matured variants and IL-6 alsorecognized the same epitope on IL-6R (FIG. 30B-C).

Example 36 Further Affinity Maturation

Next, a panel of 47 Nanobodies was generated containing differentcombinations of beneficial mutations in CDR1/2 and CDR3. These mutationswere identified by thorough analysis of protein sequences and off-ratesof all Nanobody clones isolated from the CDR randomization libraries.Off-rates of these 2^(nd) generation clones ranged from 4.2E-04 to4.5E-05 s⁻¹. 5 clones showing the slowest off-rates were selected forfurther analysis (20F6, 20A11, 20E10, 21A10, 21D11). Sequences arelisted in FIG. 31.

Example 37 Nanobody Expression and Purification of Second Round Variants

Affinity matured Nanobodies were expressed in E. coli as c-myc,(His)6-tagged proteins in a culture volume of 250 ml. Expression wasinduced by addition of 1 mM IPTG and allowed to continue for 4 h at 37°C. After spinning the cell cultures, periplasmic extracts were preparedby freeze-thawing the pellets. These extracts were used as startingmaterial for immobilized metal affinity chromatography (IMAC).Nanobodies were eluted from the column with 150 mM imidazole andsubsequently desalted on a Hiprep26/10 column.

Example 38 Determination of Melting Temperatures

Temperature stability of the Nanobodies was analyzed in the thermalshift assay. The Tm values were similar for all affinity maturedNanobodies and slightly higher as compared to IL6R65. Tm values arelisted in Table C-25.

Example 39 Affinity Determination of 2^(nd) Round Variants on Biacore

Kinetic parameters for Nanobody IL6R65 (sequence optimized) and the 5affinity matured variants were determined on Biacore T100. Associationrates (k_(a)) were determined from binding curves at 2 differentconcentrations of purified Nanobody and a fixed concentration of IL-6Rwhich was captured on the chip via mAb BN-12. Off-rates were determinedat a single Nanobody concentration using IL-6R covalently coupled to thechip, Values for ka, kd and Kd are listed in Table C-26.

Example 40 Evaluation of 2^(nd) Round Variants in TF-1 Assay

Biological activity of the 2^(nd) round variants was evaluated in theTF1 assay and data are presented in FIG. 32. The 2nd round affinitymatured clones had a 5-8 fold higher potency than 1st round clone 7C4and were 2.5-4 fold more potent than the benchmark reference IgGdescribed in Example 1. Best performing clone was 20A11 with an IC50 of0.4 nM (4-fold more potent as compared to benchmark).

Example 41 Evaluation of 2^(nd) Round Variants in Plasma Potency Assays

Inhibition of soluble IL-6R was analyzed in the plasma potency assay athigh and low IL-6 concentrations. The 2^(nd) round variants were atleast as potent as the reference IgG in the plasma potency assay(0.1-0.2 nM), which was at the sensitivity limit of this assay. At highIL-6 concentration (EC95), the affinity mutants were still able to blockbinding of IL-6 to sIL-6R and appeared to be 3-4 fold more potent thanthe reference IgG. No difference could be observed between 1st and 2ndround variants in this assay. As expected, all tested clones were cynocross-reactive (FIG. 33).

Example 42 Binding to Human PBMCs in Full Blood

Purified Nanobodies IL6R65 and PMP20A11 were tested in FACS for bindingto human PBMCs. Cell binding was detected using a biotinylated anti-HismAb and PE-labelled streptavidin (FIG. 34). Both IL6R65 and the affinitymatured variant PMP20A11 could bind to neutrophils, monocytes and asubpopulation of lymphocytes. This is in agreement with the expressionprofile of IL-6R.

VI. Formatting of Affinity Matured Nanobody Example 43 Preparation ofMultivalent Constructs

PMP20A11 was formatted as bivalent and trivalent Nanobodies with thealbumin-binding Nanobody ALBS. An overview of the different Nanobodiesis presented in Table C-27.

Example 44 Neutralization of Membrane IL-6R in the TF-1 Assay

In a first experiment, it was verified if the formatted Nanobodiesinhibit the signalization through IL-6R using the TF-1 cell line asmodel system. Nanobodies 20A11, IL6R304, IL6R305 and IL6R306dose-dependently and completely block the IL-6 induced proliferation ofTF-1 cells mediated by membrane IL-6R (FIG. 35, Table C-28).

These results demonstrate that all formatted Nanobodies are more potentcompared to the reference IgG as described in Example 1 for inhibitingmembrane IL-6R activity. Compared to its monovalent equivalent IL6R304,IL6R305 inhibits 7-fold more potently the IL-6 mediated responses,demonstrating avid interaction of IL6R305 with membrane IL-6R. IL6R306is less potent than IL6R305 and only shows a 2-fold better potency thanIL6R304. This demonstrates that the format of IL6R306 is less favorableand moreover indicates that avid binding of IL6R306 is not possible.

Next, it was verified if the formatted Nanobodies could still completelyinhibit the signalization through IL-6R at pathological concentrationsof IL-6. Indeed, 20A11, IL6R304, IL6R305 and IL6R306-dose-dependentlyand completely blocked the proliferation of TF-1 cells induced by 5000IU/mL IL-6 (FIG. 36, Table C-29). As expected, IC50s did shift comparedto the experiments performed with 100 IU/mL. In agreement with theprevious results, IL6R305 was most potent in blocking the IL-6 inducedsignalization through membrane IL-6R.

As the Nanobodies do interact with IL-6R, it was verified if binding ofNanobodies to this receptor might induce cell activation leading to cellproliferation. TF-1 cells were incubated with an excess of Nanobody inthe presence or absence of 100 IU/mL IL-6 (FIG. 37). As expected, bothIL6R304 and IL6R305 completely prevented the IL-6 mediatedproliferation. Indeed, IL6R305 and IL6R306 inhibited the IL-6proliferation to the same level as the background (³H-thymidineincorporation measured in the absence of growth factors), demonstratingthat IL6R304 and IL6R305 completely blocked the effect of IL-6.

IL6R304 and IL6R305 did not induce proliferation of TP-1 cells in theabsence of growth factors, suggesting that these compounds do not havean agonistic effect on TF-1 cells.

Example 45 Neutralization of sIL-6R in ELISA by the Formatted AffinityMatured Nanobodies

The ability of the building block IL6R20A11 and its formatted variantsto prevent binding of human IL-6 to plasma sIL-6R was analyzed in theplasma potency ELISA. Since the concentration of plasma sIL-6R isvariable, the same plasma needed to be used across the different assaysfor comparing the potency of the Nanobodies. Also, a titration of IL-6was first incubated in the plasma to determine the concentration of IL-6that would be used with the Nanobodies. The EC50 and EC95 values of IL-6in human plasma were determined to be 27.29 ng/mL and 885 ng/mL,respectively. These concentrations were subsequently used to test thepotency of the Nanobodies at normal and high concentrations of IL-6.

IL6R20A11 and the formatted variants were compared to the reference IgGas described in Example 1. The resulting IC50 values for the differentNanobodies are summarized in Table C-30. At the EC50 of IL-6 (FIG. 38A,B) both the monovalent and bivalent IL-6R Nanobodies were in the samerange as the reference IgG. Although IL6R304 has only one binding site,it had a similar IC50 as the reference IgG (0.229 nM vs. 0.258 nM).IL6R305 appeared to be twice as potent as IL6R304 (IC50 of 0.137 nM),which is in line with its two binding sites. However, IL6R306 appearedto be less potent than IL6R304 and the reference IgG and had an IC50 of0.412 nM.

Most probably the assay limit was reached in terms of sensitivity.Indeed, the concentration of plasma sIL-6R was ˜30 ng/mL or 0.6 nM.Therefore, only 0.3 nM of sIL-6R needed to be blocked by the Nanobodies(50% plasma), which means that the minimum IC50 that can be obtained is0.15 nM. This corresponds to the IC50 values that were obtained.However, if the IL-6 concentration would be increased to 885 ng/mL itwould be more difficult for the Nanobodies to compete with IL-6 and alarger difference in potency could be detected. Indeed, at high IL-6concentrations IL6R20A11, IL6R304 and 16R305 were clearly more potentthan the reference IgG, whereas IL6R306 was not (FIG. 38 C, D), Theratio of the IC50 at high and low concentrations of IL-6 is shown inTable C-30, Clearly, the reference IgG and IL6R306 were more affected byincreasing IL-6 than the other Nanobodies. This was also observed in theTF-1 assay (see Example 44).

Example 46 Binding of the Formatted Affinity Matured Nanobodies toMembrane IL-6R

In order to block signaling of IL-6, both soluble and membrane IL-6Rneed to be neutralized by the Nanobodies. Therefore, binding of thedifferent formatted Nanobodies to IL-6R-expressing cells was analyzed byflow cytometry.

Binding to IL-6R-Expressing CHO Cells

Stably transfected CHO cells expressing human IL-6R were used to analyzethe binding of the anti-IL-6R Nanobodies to membrane IL-6R (FIG. 39A).IL-6R negative CHO cells were used as a negative control (FIG. 39B). All4 Nanobodies showed saturated binding to IL-6R-expressing cells, whereasonly very low signals were detected at high Nanobody concentrations onIL-6R-negative cells.

The median PE fluorescence was exported to GraphPad and 4PL curves werefitted to determine the EC50 values. These are summarized in Table C-31.In contrast to the TF-1 assay, IL6R305 did not seem to benefit from anavidity effect in this setup: it was only a factor 2 more potent thanIL6R304 (0.8984 vs. 1.939 nM). As was also the case in the TF-1 and inthe plasma ELISA, IL6R306 bound less well to IL-6R-positive cells.

Binding to Peripheral Blood Leukocytes

Human PBL were used to demonstrate binding of the Nanobodies to membraneIL-6R under physiological conditions. This matrix is highly relevant forthe in vivo situation, since it contains HSA (˜50 mg/mL), sIL-6R (˜30rig/mL), cells expressing membrane IL-6R (CD4⁺ T cells, monocytes,granulocytes) and IL-6R-negative cells (most circulating B cells, CD8⁺ Tcells). The Nanobodies were incubated in EDTA-treated blood from 2donors and bound Nanobody was detected by flow cytometry. Lymphocytes,monocytes and granulocytes were gated based on FSC/SSC properties (FIG.40) and bound Nanobodies were detected in the PE channel.

As can be observed in FIG. 40 (right), the granulocytes and monocyteswere uniformly stained by the Nanobodies. In contrast, only a part ofthe lymphocytes were stained. This can be observed as a double peak inthe PE histogram. The mean fluorescence of the 3 gated populations afterincubation with different concentrations of the Nanobodies is depictedin FIG. 41 and the resulting EC50 values are summarized in Table C-32.

Example 47 Affinity of the Formatted Affinity Matured Nanobodies forHuman and Cyno Serum Albumin

Kinetic analysis of the bi-specific, bivalent and trivalent NanobodiesIL6R304, IL6R305 and IL6R306 on human and cyno serum albumin wasperformed by SPR on a Biacore 3000 instrument. Results are shown in FIG.42 and summarized in Table C-33. Nanobodies IL6R304, 305 and 306 showedsimilar kinetic rate constants and affinities (17-23 nM) for human andcyno SA. The affinity of the formatted IL-6R Nanobodies for SA was 6.5×lower as compared to the monovalent anti-SA Nanobody ALB11 by a decreaseof a factor 2.5 in association rate and an increase of a factor 2.5 indissociation rate.

Example 48 Affinity for Human and Cyno IL-6R

Kinetic analysis on human and cyno IL-6R was performed by SPR on aBiacore T100 instrument. Because of indications of a conformationalchange of IL-6R when immobilizing it directly to the chip on-rates weremeasured on IL-6R captured by BN-12. Off-rates were measured on directlyimmobilized IL-6R, because of the lack of availability of a capturingtool with a lower dissociation rate than the Nanobody-IL-6R interaction.Results are shown in Table C-34 and FIG. 43. The on-rate of 20A11(IL6R300; SEQ ID NO: 66) decreased by less than a factor 2 by formattingwith an anti-SA building block (IL6R304). The off-rate for IL6R304 onhuman IL-6R was at or below the detection limit of the Biacoreinstrument. The off-rate on cyno IL-6R was 2 times higher than on humanIL-6R, but still near the detection limit. Calculated affinities forIL6R304 were 14 pM on human IL-6R and 25 pM on cyno IL-6R.

Example 49 Species Cross-Reactivity of IL6R20A11

Cross-reactivity of IL6R20A11 and its formatted variants with cynomolgussIL-6R was analyzed in the plasma potency ELISA using cyno plasma. Also,a competition ELISA was used to determine the cross-reactivity ofIL6R20A11 with cyno and mouse sIL-6R.

Plasma Potency ELISA

A titration of human IL-6 was first incubated in cyno plasma and theEC50 value of IL-6 was determined to be 50.11 ng/mL. This concentrationof IL-6 was subsequently used to test the cross-reactivity of theNanobodies with cyno plasma sIL-6R. As can be observed in FIG. 44,IL6R20A11 and the formatted variants were clearly cross-reactive withcynomolgus sIL-6R. The same ranking can be observed as in human plasmaand the ratio of the IC50 values in human vs. cyno plasma was similarfor all compounds (Table C-35).

Competition ELISA

The plasma potency ELISA can only be used if BN-12 is able to captureplasma sIL-6R from that particular species and if binding of IL-6 tosIL-6R can be detected. Therefore, a more generic competition ELISA wasdeveloped. This assay was based on binding of IL6R20A11 toneutravidin-captured IL-6R-bio. Briefly, 0.4 nM of IL6R20A11 waspre-incubated with a titration series of plasma from the differentspecies containing endogenous sIL-6R, after which free IL6R20A11 wascaptured on biotinylated human sIL-6R immobilized on aneutravidin-coated plate, and detected with an anti-VHH mAb:FITC andanti-FITC-HRP. The concentration of IL6R20A11 of 0.4 nM corresponds tothe concentration that yields 50% of the maximal signal (EC₅₀ of0.35±0.021 nM; n=4).

As can be observed in FIG. 45, IL6R20A11 was clearly cross-reactive withcyno sIL-6R, which confirms the Biacore results. In contrast, no bindingto mouse sIL-6R could be observed. Human and cyno plasma also competedwith binding of IL6R20A11 to recombinant sIL-6R, whereas mouse plasmadid not. In fact, increasing signals were observed at highconcentrations of mouse plasma, which was probably due to the detectionof mouse immunoglobulins in the assay.

Example 50 Specificity of IL6R20A11 for IL-6R

IL-6R belongs to the family of type I cytokine receptors. Since thecytokine binding region of these receptors is conserved, the specificityof IL6R20A11 for IL-6R was analyzed by analyzing binding to receptorsrelated to IL-6R. Binding of IL6R20A11 to LIF-R, CNTF-R, OSM-R andIL-11R/Fc was analyzed in a competitive binding ELISA. As can beobserved in FIG. 46, the positive control sIL-6R inhibited binding ofIL6R20A11 to the plate. The IC₅₀ for sIL-6R (0.03 nM) was in line withthe expected IC50 based on the amount of IL6R20A11 that was used (0.025nM vs. 0.05 nM). None of the IL-6R-related proteins seemed to competewith binding of IL6R20A11 to sIL-6R, even at a 100-fold molar excess (5nM). In a similar setup, it was shown that CLF-1/CLC, IL12-p40, IL-27Band gp130/Fc also did not interact with 0.05 nM of IL6R20A11, even atconcentrations as high as 100 nM (results not shown).

VII. In Vivo PK/PD Analysis of IL6R304 and IL6R305

The aim of this study was to analyze the plasma pharmacokinetics (PK),pharmacodynamics (PD) and immunogenicity of two sequence optimized,affinity maturated anti-interleukin 6 receptor (IL-6R) Nanobodies,namely IL6R304 and IL6R305, in cynomolgus monkey after a singleintravenous bolus administration. Administration of Nanobodies wasfollowed by 7 daily subcutaneous injections of recombinant human (h)IL-6 starting 24 hours post Nanobody administration. The ultimate goalof this in vivo efficacy study was to assess the ability of theseanti-IL-6R Nanobodies to inhibit hIL-6-induced parameters and comparetheir efficacy with each other and with the benchmark reference ofExample 1.

In non-human primates and in humans, recombinant hIL-6 has been reportedto induce the synthesis of acute phase proteins. Acute phase proteinsare defined as a class of plasma proteins, such as C-reactive protein(CRP), serum amyloid A, haptoglobin, fibrinogen, albumin andtransferrin, whose plasma concentrations increase or decrease by atleast 25% in response to inflammation, mainly due to changes in theirproduction by hepatocytes. Patterns of cytokine production and acutephase response differ in different inflammatory conditions. Therefore,acute phase changes reflect the presence and intensity of inflammation,making them diagnostically relevant. The main stimulators of theproduction of acute phase proteins are the inflammation-associatedcytokines, which are produced during inflammatory processes: IL-6,IL-1β, tumor necrosis factor-α (TNF-α), interferon-γ (INF-γ),transforming growth factor β (TGF-β) and possibly IL-8.

Example 51 Study Design

In this study 6 groups (groups 6 to 11, Table C-36) of 2-3 animalsreceived a single i.v. injection of IL6R304 or IL6R305. Of bothNanobodies, 3 different doses were tested, namely 0.4, 2 or 10 mg/kg. Inaddition, animals in group 12 (n=3) received vehicle and served asnegative control, while animals of group 13 (n=3) were injected with 5mg/kg reference IgG (Table C-36).

Starting on TD1, i.e. 24 hours after test item administration, allanimals were injected once daily for 7 days with hIL-6 (5 μg/kg; FIG.47).

Blood samples were collected via the vena cephalica or saphena magnabefore and after injection of hIL-6 on predetermined time points (seeFIG. 47). Extra blood samples were taken on TDO for PK analysis (seeTable C-37).

Example 52 The Effect of the Nanobodies on an hIL-6-Induced Acute PhaseResponse (Phase 2)

The effect of the Nanobodies and the positive reference IgG was analyzedon the induction of an acute phase response by 7 consecutive dailyinjections of hIL-6. Read-outs were CRP levels, fibrinogen levels andplatelet count.

In the negative control group (group 12), CRP levels were immediatelyelevated after the first injection of hIL-6 and maximum levels werealready reached on day 2. The maximum levels attained were between0.2-0.8 mg/mL and this plateau was maintained until day 8, which is theday after the last hIL-6 injection (FIG. 48A).

These changes were completely inhibited by pretreatment with 5 mg/kgreference IgG (FIG. 48B). Also a single pretreatment with a comparabledose (2 mg/kg) or a 5-fold higher dose (10 mg/kg) of the NanobodiesIL6R304 and IL6R305 gave almost complete inhibition of CRP inductionduring the whole course of the experiment (FIGS. 48C and D). Only animalNo. 18 in the highest dose group of IL6R304 showed some induction,reaching maximum serum CRP levels around 0.1 mg/mL. In the lowest dosegroup (0.4 mg/kg), both Nanobodies gave complete inhibition for 7 days.CRP levels increased only on day 8 to comparable levels as in thenegative control group (FIG. 48) The mean CRP levels of all groups canbe found in FIG. 49.

Fibrinogen levels increased slowly in the negative control group to anaverage maximum of 5 times the basal levels (FIG. 50A). This maximum wasreached on day 6 and was maintained for 2 more days. On day 14,fibrinogen levels were back to basal levels. The reference IgG wascapable to completely inhibit the induction of fibrinogen (FIG. 50B).Both Nanobodies showed a dose-dependent inhibition of fibrinogeninduction (FIGS. 50C and D). In the highest dose groups, inhibition wasalmost complete for both animals pretreated with IL6R304 and for 1 outof 2 animals pretreated with IL6R305. There was some minor increases infibrinogen levels of animal No. 25 (10 mg/kg IL6R305) and of all animalsof the middle and lowest dose groups of both Nanobodies. In theseanimals, however, fibrinogen levels never exceeded 2-3 times the basallevels. The mean fibrinogen levels of all groups can be found in FIG.51.

For all animals in the negative control group, platelet counts increasedslowly from day 5 onwards. Maximum levels were reached at day 8 to day14 and were between 160-190% of basal levels. The effect of hIL-6 onplatelet counts was completely blocked by a single pretreatment with 5mg/kg reference IgG or 2 mg/kg of the Nanobodies. An induction inplatelet count was only observed in the lowest dose groups of theNanobodies, starting in all animals on day 8. Maximal induction wasaround 120-150% of basal levels for IL6R304, while maximum plateletcounts for IL6R305 were between 160-180% of basal levels (FIGS. 52 and53).

In conclusion, IL6R304 and IL6R305 showed a similar dose-dependent andcomplete inhibition of all three acute phase response parameters.

Example 53 Plasma Concentrations After i.v. Administration of IL6R304 orIL6R305 (0.4-2-10 mg/kg) in Cynomolgus Monkeys

Blood samples for plasma pharmacokinetic (PK) analysis ELISA sampleanalysis were taken at pre-dose and at following time points postadministration of IL6R304 or IL6R305: 5 and 30 minutes, 3 and 8 hours,day 1, 2, 3, 4, 5, 6, 7, 8, 14, 21 and 29.

Individual observed plasma concentration-time plots after i.v.administration of IL6R304 (0.4-2-10 mg/kg) and IL6R305 (0.4-2-10 mg/kg)to cynomolgus monkeys are shown in FIG. 54 and FIG. 55, respectively.

Example 54 Non-Compartmental Pharmacokinetics Analysis of IL6R304 andIL6R305 (0.4-2-10 mg/kg) in Cynomolgus Monkeys

An overview of the basic PK parameters obtained by non-compartmentalPI<analysis of IL6R304 (0.4-2-10 mg/kg) and IL6R305 (0.4-2-10 mg/kg) incynomolgus monkeys is given in Table C-38, Table C-39, Table C-40, TableC-41, Table C-42 and Table C-43. The PK parameters discussed herein areobtained using non-compartmental analysis (NCA) using WinNonlinProfessional Software Version 5.1 (Pharsight Corp). The terminalparameters for some of the animals were calculated with two data-pointsonly (R² is 1 by default).

For both Nanobodies administered intravenously in cynomolgus monkeys,plasma concentrations seemed to decline in a triphasic manner. Duringthe first two days post administration there was an initial dispositionphase, followed by a slower dominant phase. A gradual decline at thelower concentrations resulted in a terminal phase characterized by ashort half-life.

Since anti-drug antibodies were detected in the plasma samples of mostcynomolgus monkeys, the change in terminal half-life at lowerconcentrations could be linked to an immune-mediated clearancemechanism. This is however unlikely upon examining the PK profiles: atthe lowest dose the shortest half-life has been observed at time pointswhere no immunogenicity is detected. Moreover, despite the presence ofdetectable titers at the higher doses, there is still a tendency towardslonger half-lives (f.e. IL6R304 10 mg/kg i.v.).

Based on PK profile observations, it is expected that both Nanobodiesare cleared from the circulation via at least two mechanisms. In such asituation, a linear non-saturable clearance mechanism would representthe non-specific degradation of compound. A second saturable clearancemechanism would be target mediated (f.e. internalization of drug bindingto membrane bound IL-6R and subsequent clearance). At higher Nanobodyconcentrations, the latter clearance mechanism is expected to besaturated and negligible compared with the non-saturable linearclearance: the linear clearance is dominant (resulting in a dominanthalf-life). However, at lower concentrations the rate of metabolism ishigher for a given Nanobody concentration, resulting in a change ofterminal slope.

Because of target mediated clearance, PK parameters obtained via NCAanalysis such as clearance and half-life appear to be dose and timedependent. The total clearance is the highest at the lowest dose: 24.8and 35 mL/day/kg for IL6R304 and IL6R305 after 0.4 mg/kg i.v. comparedwith 10.4-9.00 and 5.93-7.76 mL/day/kg for IL6R304 and IL6R305 at thehigher doses. Correspondingly, the dose normalized exposure will belower at the lowest dose (Dose=CL×AUC).

The dominant half-life of IL6R304 decreased from 6.61 days to 5.00 daysand 1.73 days after i.v. administration of 10, 2 and 0.4 mg/kg. Thedominant half-life of IL6R305 decreased from 7.37 days to 4.29 days and1.64 days after i.v. administration of 10, 2 and 0.4 mg/kg. Since moredata points are available at earlier time points, the terminal phase wasbest characterized at the lowest dose: a short terminal half-life of0.530 days and 0.470 days was observed after ix. administration of 0.4mg/kg IL6R304 and IL6R305, respectively.

Based on these PK findings, the pharmacokinetic properties of IL6R304and 116R305 are considered to be similar.

At test day 29 for monkey 14m and monkey 15f (i.v. 2 mg/kg) low 116R304concentration levels were still detectable. Based on the PK profiles ofthe other monkeys, these observations were unexpected. It is possiblethat this indicates a second type of saturable target binding, whichonly becomes apparent at very low concentrations. However, theseobservations could also be an artifact of the PK ELISA sample analysis.

The reported volumes of distribution calculated via NCA analysis werelow ranging from once to twice the plasma volume of approximately 40mL/kg for both Nanobodies, suggesting limited distribution outside thevascular space, However, the true Vss may be underestimated due tomethodological errors linked to NCA (f.e. Nanobody distribution andsubsequent degradation in the peripheric space would not be attributedto the distribution term but to the total systemic clearance). The Vssseems fairly constant across the different dose levels.

To illustrate the possible effects of target binding on the PK profile,FIG. 56 compares the mean PK profiles of IL6R304 (i.v. 0.4-2-10 mg/kg)and IL6R202 (i.v. 2 mg/kg, SEQ ID ON: 73). For illustrative purposes theIL6R202 (i.v. 2 mg/kg) PK profile was normalized to a 0.4 mg/kg and 10mg/kg dose as well. IL6R304 was shown to bind to IL-6R in cynomolgusmonkey, while for IL6R202 there was a lack of target binding incynomolgus monkey (WO 09/010,539). Correspondingly, at highconcentrations where linear clearance is dominant, the profiles (andcorresponding half-lives) of both Nanobodies are similar. At lowerconcentrations, a gradual change in slope is observed for IL6R304 mostlikely due to target mediated clearance, while for IL6R202 there is nochange in terminal slope. However, the lower concentrations of 11.68304and IL6R305 could also be somewhat underestimated due to IL-6Rinterference in the PK ELISA, especially if high IL-6R levels arepresent.

Example 55 Detection of Anti-IL6R304 and Anti-IL6R305 Antibodies

A series of plasma samples taken at predose and at different days postadministration of IL6R304 or IL6R305 were screened for the presence ofmonkey antibodies (IgG isotype) capable of binding to the Nanobody orone or more of its building blocks. Samples from animals dosed withIL6R304 were analyzed on plates coated with either IL6R304 (FIG. 57),IL6R300 (FIG. 58) or ALBS (FIG. 59). Samples from animals dosed withIL6R305 were analyzed on plates which were coated with either IL6R305(FIG. 60), IL6R300 (FIG. 61) or ALBS (FIG. 62).

A summary of the anti-drug antibody (ADA) appearance to full Nanobody(IL6R304 and IL6R305) is given in Table C-44. Lower or no response wasobserved to IL6R300 and Alb8. In conclusion, after i.v. injection ofIL6R304, ADA were detectable in all monkeys (except for Animal No. 16 inwhich no ADA determination could be determined due to high predosevalues). Antibodies appeared after 1 week post administration for themonkeys dosed at 0.4 mg/kg and after 2 weeks post administration for themonkeys dosed at 2 mg/kg and 10 mg/kg. Highest ADA titers were obtainedin animals No. 11 and 13 (both from the 0.4 mg/kg dose). After i.v.injection of IL6R305, ADA were detectable in all monkeys (except foranimal No. 23 in which no ADA was detected). Antibodies appeared after 1week post administration for the monkeys dosed at 0.4 mg/kg and after 2weeks post administration for the monkeys dosed at 2 mg/kg and 10 mg/kg.Highest ADA titers were obtained in animals No. 22 and 24 (both from the2 mg/kg dose) and in animals No. 25 and 26 (both from the 10 mg/kgdose).

Example 56 The Effect of the Nanobodies on sIL-6R Levels

As expected based on publicly available data (Nishimoto et al., 2008,Blood 112(10): 3959-64), treatment with Ref IgG lead to an increase inplasma sIL-6R, whereas treatment with vehicle did not (FIG. 63A).Similarly, the low dose of IL6R304 induced a rapid increase in sIL-6Rlevels in all three monkeys (FIG. 63B). In the IL6R305-treated animalsplasma sIL-6R also increased, but not as pronounced as in the othertreatment groups (FIG. 63C).

VIII. Efficacy of IL6R304 Example 57 The Effect of the Nanobodies onsIL-6R Levels in the Efficacy Study

The total sIL-6R levels in plasma (free, Nanobody-bound and IL-6-bound)were measured via ELISA. As expected based on published data (Nishimotoet al., 2008, Blood 112(10): 3959-64), treatment with Ref IgG led to anincrease in plasma sIL-6R, whereas treatment with vehicle did not (FIG.64A). Similarly, all Nanobody-treated animals showed a rapid increase insIL-6R levels. This can be explained by a slower clearance of the RefIgG- or NB-sIL-6R complex compared to free sIL-6R.

The maximum sIL-6R level and the duration of the effect were clearlydose dependent. Also, the effect of the Nanobodies seems to be morepronounced than for the Ref IgG (compare the 2 mg/kg dose of theNanobodies to Ref IgG in FIG. 64). This can possibly be explained due tofaster Fc-mediated clearing of antibody immune complexes. Surprisinglyhowever, the increase in sIL-6R and the duration of the effect were morepronounced for IL6R304 (FIG. 64A) compared to IL6R305 (FIG. 64B).

Example 58 The Effect of the Nanobodies on IL-6 Levels in the EfficacyStudy

The total IL-6 levels in plasma (free, sIL-6R-bound) were measured viathe Gyrolab platform. For this assay, a biotinylated rat anti-human IL-6mAb was used to capture IL-6 and an Alexa-labeled mouse anti-human IL-6mAb for detection. The assay measures both endogenous cynomolgus monkeyIL-6 and recombinant human IL-6 that is injected daily from days 1-8.Therefore, a distinction needs to be made between the IL-6 that can bemeasured until day 1 (=Only endogenous cyno IL-6) and from days 2-29(=administered human IL-6+endogenous cyno IL-6).

As can be observed in FIG. 65, administration of IL6R304, IL6R305, RefIgG and to a minor extent placebo, led to a transient increase in IL-6which peaked at 8 h post administration. However, this increase wasmarkedly higher in the Nanobody-treated groups. The effect on IL-6 didnot seem to be specific for IL-6R targeting Nanobodies, sinceadministration of an irrelevant Nanobody also led to a transient IL-6response (FIG. 650). The early increase in IL-6 can most probably beexplained by stress because of handling of the animals and potentially afurther increase in IL-6 production by minute amounts of endotoxins inthe Nanobody preparations.

During the IL-6 treatment phase, blood was sampled before each dailyinjection of IL-6. In the placebo group, which received IL-6 but not theNanobodies or Ref IgG, almost no IL-6 was detected (FIG. 65C). This isin line with the short half life of human IL-6 after SC injection(Tsigos, et al., 1997, J. Clin. Enclocrinol. Metab. 82: 4167-70).However, in all animals treated with 2-10 mg/kg Ref IgG, IL6R304 orIL6R305, IL-6 was detected from days 2-8. This can be explained byblocking of receptor-mediated clearance of IL-6, thereby prolonging itshalf life (Nishimoto et al., 2008, Blood 112(10): 3959-64). Hence,circulating IL-6 could serve as a pharmacodynamic biomarker forneutralization of IL-6R.

IX. Pharmacodynamics of IL6R304 Example 59 Pharmacodynamic Effects AfterSingle Dosing in Cynomolgus Monkey

Changes in sIL6R plasma concentrations were also measured after singlei.v. administration of 116R304 in healthy (i.e. non-stimulated)cynomolgus monkeys. In this single dose PK/PD study, doses ranged from1-100 mg/kg. Blood sampling was performed for pharmacokinetic,immunogenicity and pharmacodynamic analysis. An ELISA-based assay wasused to measure total sIL6R levels and a ligand binding assay using theGyrolab™ platform was used to measure free sIL6R levels. For the totalsIL6R assay, a non-neutralizing anti-IL6R monoclonal antibody was usedto capture sIL6R (free + in complex) and a polyclonal biotinylatedanti-IL6R tool in combination with streptavidin-HRP for detection. Forthe free sIL6R assay, the biotinylated 20A31 building block was used tocapture the free sIL6R and an Alexa-labeled non-neutralizing anti-IL6Rmonoclonal antibody for detection.

The results of the single dose PK/PD study confirmed the dose-dependenteffect of IL6R304 on (I) the maximal total sIL6R concentrations (FIG.66A), (ii) the duration of (increased total sIL6R (FIG. 66A) and (iii)the duration of the suppression of free sIL6R (FIG. 668). In general,total and free sIL6R concentrations returned to baseline levels after agiven time (dependent on the dose, longest elevation and suppression isseen with highest dose). Concentrations of free sIL6R afteradministration of a low dose of 1 mg/kg IL6R304 were decreased forapproximately 8 days and then increased to concentrations above those ofvehicle treated animals.

A good inverse correlation was observed between total sIL6R levels, freesIL6R levels (PD) and IL6R304 concentrations confirming that sIL6R canbe used as a biomarker for the presence of active drug. FIG. 67 showsone example to elucidate the interplay between the 3 parameters (the middose group was selected as the return to baseline can be demonstrated inthis group): administration of IL6R304 lead to an increase in totalsIL6R concentrations and a stabilized drug-sIL6R complex was formed. AsIL6R304 plasma concentrations decreased, concentrations of total sIL6Ralso decreased in parallel as most of the total sIL6R constituted ofcomplexed receptor. This is confirmed by low free sIL6R concentrationswhich return to baseline levels upon elimination of IL6R304 from thecirculation. Low free sIL6R concentrations confirm that measured totalsIL6R is indeed inactive and complexed with IL6R304 (FIG. 67).

Example 60 Description of Pharmacodynamic Effects Via PK/PD Modeling

The influence of IL6R304 administration on total sIL6R levels can beexplained by direct binding of IL6R304 to the receptor—the complex staysin circulation via the half-life extension moiety of IL6R304 (i.e.albumin binding). As the measurable changes in total sIL6Rconcentrations follow a time-delayed kinetic, an indirect response modelbest describes the PK/PD relationship and was used to describe theeffect of i.v. administered IL6R304 on the accumulation of sIL6R-IL6R304complex levels. The model describes a drug response that results fromthe inhibition of the elimination of sIL6R when bound to IL6R304. Inthis indirect response model, the rate of change of total sIL6R-IL6R304complex (Response R) is described by:

$\frac{R}{t} = {{Kin} - {{Kout}*\left\lbrack {1 - {I\; \max*\frac{C^{n}}{{{IC}\; 50^{n}} + C^{n}}}} \right\rbrack*R}}$

With K_(in), the zero order synthesis rate; R, the total sIL6R; I_(max),the maximum inhibition; C, the IL6R304 plasma concentration; n, thedose-response shape factor; and K_(out), the first order eliminationrate constant of sIL6R.

All available i.v. total sIL6R data from the single dose PK/PD studywere fitted simultaneously to the model (WinNonlin Professional SoftwareVersion 5.1, Pharsight Corporation, Mountain View Calif., USA) using thepharmacokinetic function as described in Example 61 as input functionfor the indirect response PK/PD model.

FIG. 68 shows the individual observed and model-predicted total sIL6Rconcentration-time data in cynomolgus monkey after i.v. administrationof 1, 5, 10, 25 or 100 mg/kg. The estimated pharmacodynamic parametersof IL6R304, in cynomolgus monkey, are listed in Table C-45. Allparameters were estimated with a sufficient degree of precision asindicated by CV % values below 50%.

All data after i.v. administration, from the single dose PK/PD study,were fitted simultaneously to an indirect response model describing thebehavior of sIL6R, IL6R304 and the complex of sIL6R-IL6R304.

The average half-life of sIL6R was estimated to be approximately 5.8 h(=ln 2/K_(out) with K_(out)=K_(n)/R₀) and an estimated production rateof 2.49 ng/mL/h. IL6R304 was able to almost completely inhibit theelimination of sIL6R via the primary pathway (I_(max)=97%). Thereforethe elimination rate changed to a new maximum decreased k_(out) whichcorrelated with that of cynomolgus monkey serum albumin. Subsequently anew baseline level of total sIL6R was established. With an estimatedIC₅₀ of 125 ng/mL or 4.48 nM, IL6R304 was shown to be a potent inhibitorof the elimination of non-complexed sIL6R in cynomolgus monkey.

X. Pharmacokinetics of IL6R304 Example 61 Pharmacokinetics in theCynomolgus Monkey

This section summarizes data characterizing the pharmacokinetic behaviorof i.v. administered IL6R304 in 1 cross-reactive species (cynomolgusmonkey).

In healthy (non-induced) cynomolgus monkeys, the concentrations ofIL6R304 were measured using a qualified DELFIA (dissociation enhancedlanthanide fluoro-immunoassay) method. Total active IL6R304concentrations were measured by means of an IL6R dependent assay.

In a single dose PK/PD study, IL6R304 was administered to healthy malecynomolgus monkeys as a single i.v. bolus of 0, 1, 5, 10, 25 and 100mg/kg. Blood samples for PK, ADA (anti-drug antibodies) and PD analysispurposes were collected from all animals at predose and at selected timepoints postdose. Samples were analysed for PK, PD and ADA (see alsoExample 62).

A validated electrochemiluminescent (ECL) bridging screening andconfirmation assay was used to detect anti-IL6R304 antibodies. Briefly,IL6R304 was used to capture and detect anti-drug antibodies (ADAs) in anhomogenous assay format using a MSD Sector Imager 2400.

For the PK analysis, IL6R304 was captured via a biotinylatedanti-Nanobody tool (3E8biv-bio) on streptavidin coated plates. After acomplexation step with the target IL6R, an Europium-labeled mAb againstIL6R was used to generate a fluorescence signal in enhancement solution.

The mean plasma concentration-time profiles of IL6R304 are displayed inFIG. 69. A summary of the key pharmacokinetic parameters of IL6R304,after a single i.v. bolus of 1, 5, 10, 25 or 100 mg/kg, are provided inTable C-46.

The pharmacokinetic profile, after i.v. administration, showed atriphasic decline. During the first two post administration days, adistribution phase was observed followed by a slower dominant phase anda faster terminal phase. The distribution phase can be further dividedin a fast (shallow compartment) and a slow distribution (deepcompartment). Based on the elimination phase, IL6R304 is presumablycleared via two mechanisms, a linear non-saturable (non-specificelimination or CL_(NON-IL6R)) and a non-linear saturable (targetmediated or specific elimination or CL_(IL6R)) clearance mechanism. Thelatter could be the result of the internalization of IL6R304 bound tomembrane bound IL6R and subsequent clearance of the IL6R304-mIL6Rcomplex.

As the clearance of IL6R304 is a combination of saturable andnon-saturable pathways, the plasma kinetics in cynomolgus monkey showeda non-linear behavior with a half-life which is dose-dependent and at agiven dose level, also time-dependent.

When the CL_(IL6R) is saturated, and the overall CL is mainly determinedby CL_(NON-IL6R), the reported half-life of IL6R304 in cynomolgus monkeyranged from 5.8 to 8.9 days and was similar to that reported forcynomolgus monkey serum albumin (Nguyen et al., 2006, Protein Eng. Des.Sel. 19: 291). This was in line with expectations and with the confirmedcross-reactivity of IL6R304's albumin binding moiety to cynomolgusmonkey albumin.

Average exposures, after single dose administration, increased somewhatmore than dose-proportional between 1 and 5 mg/kg and 10 and 25 mg/kgand dose-proportional between 5 and 10 mg/kg. The result of the 100mg/kg dose group has to be taken with caution as only one animal wasincluded in this dose group. Overall, due to the limited number ofmonkeys per dose group, the dose-proportionality assessment wasexploratory.

The binding of IL6R304 to sIL6R resulted in an increase of the measuredtotal concentration of sIL6R, which comprised of sIL6R and sIL6R-IL6R304complex; this increase is thought to be due to a slower clearance of thecomplex compared to the sIL6R alone.

Based on the available immunogenicity, PK and PD data, it was concludedthat emerging RDAs likely have impacted the PK/PD profile of IL6R304 intwo animals from the highest dose group (animal 15 and 17). Both animalsshowed an unexpected decrease in IL6R304 plasma levels concurrent to theemergence of measurable ADA and with a reduced pharmacodynamic effect.Therefore, these animals were not considered in the PK/PD analysis.Emerging ADAs in the other animals did not seem to have an obviouseffect on the PK/PD profiles, therefore these data were included in theanalysis.

For one animal (animal 3) in the 1 mg/kg dose group, no target mediatedclearance was observed, although this was expected for this low dose.For one animal (animal 6) in the 5 mg/kg dose group, target mediatedclearance was still observed despite the higher dose and expectedsaturation of this pathway. As the variability for endogenous IL6Rconcentrations between animals can be high, the target mediatedclearance can be subjected to a high inter-individual variability.Additionally, when the IL6R304 concentrations are close to the estimatedKM value (here: 0.718 μg/mL), a small change in the IL6R304concentration results in a large change in nonlinear clearance. Thecombination of both can lead to a measurable evidence of target or onlynon-target mediated clearance in the lower dose groups which isreflected in a high variability in terminal half-life values. The PKparameters of the animals 3 and 6 were excluded from descriptivestatistics as the biologic variability excludes meaningful assessment ofthe precision of applied methods (Table C-46).

None of the animals from the placebo group were systematically exposedto IL6R304. All predose samples of the IL6R304 treated animals werebelow the lower limit of quantification (LLOQ). Animals from the activetreated groups, showed an increase in plasma concentrations of IL6R304with an increase in dose level. The highest mean total exposure(AUC_(inf)) was observed in the highest dose group (100 mg/kg) and was540612 μg·h/mL.

Mean dose-normalized AUC_(inf) values increased dose proportionally overthe 5 to 10 mg/kg dose range and somewhat more than dose proportionalbetween 1 and 5 mg/kg and 10 and 25 mg/kg (1.3 and 1.4, respectively).The more than dose-proportional increase from 25 to 100 mg/kg has to betaken with caution as only one animal was included in the highest dosegroup. Overall, due to the limited number of monkeys per dose group, thedose-proportionality assessment was exploratory.

Notably, data from the non-compartmental analysis indicated a differencein half-life at the 1 mg/kg dose level compared to higher dose levelstested. This is attributable to the higher contribution of saturatabletarget mediated clearance mechanisms as compared to higher doses wherenon-saturable mechanisms prevail.

Based on the elimination phase, IL6R304 is presumably cleared via twomechanisms, a linear and a non-linear clearance mechanism. The linearclearance mechanism is likely related to the non-saturable, and non-IL6Rmediated removal of IL6R304 and corresponds to the slow and non-specificproteolytic degradation of IL6R304. The non-linear and IL6R-mediatedclearance process is a saturable clearance mechanism; most probablyrepresenting binding of IL6R304 to membrane bound IL6R and subsequentinternalization and clearance.

The non-linear pharmacokinetic behavior of IL6R304 in the cynomolgusmonkey was captured by fitting the data to an open three-compartmentalpharmacokinetic model with linear and a non-linear clearance from thecentral compartment. The structural model is depicted in FIG. 70.

All available individual i.v. plasma concentration data from a singledose PK/PD study were fitted simultaneously to the model (WinNonlinProfessional Software Version 5.1 (Pharsight Corporation, Mountain ViewCalif., USA) using iterative re-weighting (1/ŷ*ŷ), where ŷ is thepredicted plasma concentration. FIG. 71 displays the individual observedand model-predicted plasma concentration-time plots of IL6R304 incynomolgus monkey after i.v. administration of 1, 5, 10, 25 or 100mg/kg. The estimated pharmacokinetic parameters of IL6R304 are listed inTable C-47.

All data from single dose PK/PD study, were fitted simultaneously to anopen three-compartmental model with linear (CL_(NON-IL6R)) andnon-linear (CL_(IL6R)) clearance from the central compartment. At lowIL6R304 concentrations (C<<<K_(m)) the contribution of the IL6R-mediatedclearance (CL_(IL6R)) is predominant and equals V_(max)/K_(m). At highIL6R304 concentrations (C>>>K_(m)), the IL6R-mediated clearance pathwaybecomes saturated and will proceed at the maximum mass turnover (i.e.V_(max)). Consequently, the overall clearance (CL) is dominated by thelinear, non-IL6R mediated pathway (CL_(NON-IL6R)).

The non-linear IL6R mediated component in the clearance explains boththe time- and dose-dependency in the half-life of IL6R304 in cynomolgusmonkey.

TABLE A-1 Preferred combinations of CDR sequences SEQ SEQ SEQ SEQ SEQSEQ SEQ SEQ Nanobody ID FR1 ID CDR 1 ID FR2 ID CDR 2 ID FR3 ID CDR 3 IDFR4 ID PMP7F4 60 EVQLVESGGGLVQPG 74 VNVMA 81 WYRQAPG 83 GIINGGST 90RFTISRDNAKNTLYLQ 92 VTTNSDY 95 WGQGT 96 GSLRLSCAASGTTFK KGRELVA TYADSVKGMNSLRPEDTAVYYCAF DLGRDY LVTVSS PMP7C4 61 EVQLVESGGGLVQPG 75 INVMA 81WYRQAPG 83 GIITNGST 85 RFTISRDNAKNTLYLQ 92 VTTNSDY 95 WGQGT 96GSLRLSCAASGTTFR KGRELVA SYADSVKG MNSLRPEDTAVYYCAF DLGRDY LVTVSS PMP7D662 EVQLVESGGGLVQPG 76 VNVMA 81 WYRQAPG 83 AVINGGTT 86 RFTISRDNAKNTLYLQ92 VTTNSDY 95 WGQGT 96 GSLRLSCAASGSIFR KGRELVA TYADSVKG MNSLRPEDTAVYYCAFDLGRDY LVTVSS PMP7G7 63 EVQLVESGGGLVQPG 74 INIMA 82 WYRQAPG 83 GVITGGNT87 RFTISRDNAKNTLYLQ 92 VTTNSDY 95 WGQGT 96 GSLRLSCAASGTTFK KGRELVATYADSVKG MNSLRPEDTAVYYCAF DLGRDY LVFVSS PMP7G8 64 EVQLVESGGGLVQPG 77INVMA 80 WYRQAPG 83 GVINDGST 88 RFTISRDNAKNTLYLQ 92 VTTNSDY 95 WGQGT 96GSLRLSCAASGSTFR KGRELVA TYADSVKG MNSLRPEDTAVYYCAF DLGRDY LVTVSS PMP20F665 EVQLVESGGGLVQPG 78 INVMA 80 WYRQAPG 83 GIVSGGST 89 RFTISRDNAKNTLYLQ92 ITTNSDY 94 WGQGT 96 GSLRLSCAASGSVFK KGRELVA SYADSVKG MNSLRPEDTAVYYCAFDLGRRY LVTVSS PMP20A11 66 EVQLVESGGGLVQPG 78 INVMA 80 WYRQAPG 83GIISGGST 84 RFTISRDNAKNTLYLQ 92 ITTESDY 93 WGQGT 96 GSLRLSCAASGSVFKKGRELVA SYADSVKG MNSLRPEDTAVYYCAF DLGRRY LVTVSS PMP20E10 67EVQLVESGGGLVQPG 78 INVMA 80 WYRQAPG 83 GIVSGGST 89 RFTISRDNAKNTLYLQ 92ITTESDY 93 WGQGT 96 GSLRLSCAASGSVFK KGRELVA SYADSVKG MNSLRPEDTAVYYCAFDLGRRY LVTVSS PMP21A10 68 EVQLVESGGGLVQPG   79 INVMA 80 WYRQAPG 83GIVTGGST 91 RFTISRDNAKNTLYLQ 92 ITTESDY 93 WGQGT 96 GSLRLSCAASGSIFKKGRELVA SYADSVKG MNSLRPEDTAVYYCAF DLGRRY LVTVSS PMP21D11 69EVQLVESGGGLVQPG 78 INVMA 80 WYRQAPG 83 GIVTGGST 91 RFTISRDNAKNTLYLQ 92ITTESDY 93 WGQGT 96 GSLRLSCAASGSVFK KGRELVA SYADSVKG MNSLRPEDTAVYYCAFDLGRRY LVTVSS

TABLE B-1 Amino acid sequences that make up the reference compoundsREFERENCE IGG HEAVY CHAIN, SEQ ID NO: 1QVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKREFERENCE IGG LIGHT CHAIN, SEQ ID NO: 2DIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC REFERENCE FAB HEAVY CHAIN, SEQ ID NO: 3QVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC REFERENCE FAB LIGHT CHAIN, SEQ ID NO: 4DIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

TABLE B-2 Protein sequences of anti-IL-6R NanobodiesPMP40H5, SEQ ID NO: 5EVQLVESGGGLVQPGGSLRLSCAASGFSLDYYAIGWFRQAPGKEREGVSCMDSSSGTTSTYYSDSVKGRFTISRDDAKNTVYLQMNSLKPEDTAVYYCAADGHLNWGQRYVPCSQISWRGWNDYWGQGTQVTVSS PMP35E11, SEQ ID NO: 6EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYAIGWFRQAPGKEHEGVSCISSSDGSTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAAERDVPARSLCGSYYWYDYRGQGTQVTVSSPMP32C9/IL6R04, SEQ ID NO.: 7EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYDIGWFRQAPGKEREGVSGISSSDGNTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTQVTVSSPMP35H4/IL6R13, SEQ ID NO: 8EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRATEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTQVTVSSPMP32E10, SEQ ID NO: 9EVQLVESGGGLVQPGGSLRLSCAASGFTFGSYDMSWVRQAPGKGPEWVSAINSGGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCATDWRYSDYDLPLPPPGDYWGQGTQVTVSSPMP30C11, SEQ ID NO: 10EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYDMGWYRQAPGKEREFVAVISRSGSSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCKAEVVAGDYDYWGQGTQVTVSSPMP35C10, SEQ ID NO: 11EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYDMGWYRQAPGKEREFVAVIHWSSGSTYYADPVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCNAFLPGPEGFHDYWGQGTQVTVSSPMP34G9, SEQ ID NO: 12EVQLVESGGGLVQAGGSLRLSCAASGRTSSSYDMTWYRQVPGKEREFVAVISWSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCNAYTGGGDDYWGQGTQVTVSSPMP31A4/IL6R03, SEQ ID NO: 13EVQLVESGGGLVQAGGSLRLSCAASGSIFKVNAMGWYRQAPGKQRELVAGIISGGSTNYADSVKGRLTISRDNAKNTVYLQMNSLKPEDTAVYYCSFVTTNSDYDLGRDYWGQGTQVTVSSPMP32E2, SEQ ID NO: 14EVQLVESGGGLVQAGGSLRLSCAASGNIFDDNTMGWTWNRQPPGKQRELVAIIATDGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLFSLRLGRDYWGQGTQVTVSSPMP33A3, SEQ ID NO: 15EVQLVESGGGLVQPGGSLRLSCAASGFTLDYGAIGWFRQAPGKEREGVSCISSSTGSTYYADSVKGRFTISRDNGKNTVYLQMNSLKPEDTAVYYCAADKMWSPCLVAANEEALFEYDYWGQGTQVTVSSPMP34A12, SEQ ID NO: 16EVQLVESGGGLVQPGGSLRLSCVASGFSLDYYVIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLLRTPEFCVDSAPYDYWGQGTQVTVSSPMP28E11, SEQ ID NO: 17EVQLVESGGGLVQPGGSLRLSCAASGFPLDYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCALVHTTAQATGVPQREYEYEWWGQGTQVTVSSPMP35F4, SEQ ID NO: 18EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYDMGWYRQAPGKEREFVAIITWNSSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCNAQYGLGYAEDYWGQGTQVTVSS

TABLE B-3 Protein sequences of multivalent anti-IL6R NanobodiesIL6R22, SEQ ID NO: 19EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYDMGWYRQAPGKEREFVAVISRSGSSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCKAEVVAGDYDYWGQGTQVTVSSGGGGSGGGSAVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSIL6R23, SEQ ID NO: 20EVQLVESGGGLVQAGGSLRLSCAASGSIFKVNAMGWYRQAPGKQRELVAGIISGGSTNYADSVKGRLTISRDNAKNTVYLQMNSLKPEDTAVYYCSFVTTNSDYDLGRDYWGQGTQVTVSSGGGGSGGGSAVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSIL6R24, SEQ ID NO: 21EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYDIGWFRQAPGKEREGVSGISSSDGNTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTQVTVSSGGGGSGGGSAVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSIL6R25, SEQ ID NO: 22EVQLVESGGSLVQPGGSLRLSCAASGFTFGSYDMSWVRQAPGKGPEWVSAINSGGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCATDWRYSDYDLPLPPPGDYWGQGTQVTVSSGGGGSGGGSAVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSIL6R26, SEQ ID NO: 23EVQLVESGGGLVQAGGSLRLSCAASGNIFDDNTMGWTWNRQPPGKQRELVAIIATDGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLFSLRLGRDYWGQGTQVTVSSGGGGSGGGSAVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSIL6R28, SEQ ID NO: 24EVQLVESGGGLVQPGGSLRLSCVASGFSLDYYVIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLLRTPEFCVDSAPYDYWGQGTQVTVSSGGGGSGGGSAVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTV SSIL6R29, SEQ ID NO: 25EVQLVESGGGLVQAGGSLRLSCAASGRTSSSYDMTWYRQVPGKEREFVAVISWSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCNAYTGGGDDYWGQGTQVTVSSGGGGSGGGSAVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRETISRDNAKTTLYLQMNSLFPEDTAVYYCTIGGSLSRSSQGTQVTVSSIL6R30, SEQ ID NO: 26EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYDMGWYRQAPGKEREFVAVIHWSSGSTYYADPVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCNAFLPGPEGFHDYWGQGTQVTVSSGGGGSGGGSAVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSIL6R31, SEQ ID NO: 27EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYAIGWFRQAPGKEHEGVSCISSSDGSTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAAERDVPARSLCGSYYWYDYRGQGTQVTVSSGGGGSGGGSAVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSIL6R32, SEQ ID NO: 28EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYDMGWYRQAPGKEREFVAIITWNSSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCNAQYGLGYAEDYWGQGTQVTVSSGGGGSGGGSAVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSIL6R33, SEQ ID NO: 29EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRATEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTQVTVSSGGGGSGGGSAVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSIL6R34, SEQ ID NO: 30EVQLVESGGGLVQPGGSLRLSCAASGFSLDYYAIGWFRQAPGKEREGVSCMDSSSGTTSTYYSDSVKGRFTISRDDAKNTVYLQMNSLKPEDTAVYYCAADGHLNWGQRYVPCSQISWRGWNDYWGQGTQVTVSSGGGGSGGGSAVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS IL6R43, SEQ ID NO: 31EVQLVESGGGLVQAGGSLRLSCAASGSIFKVNAMGWYRQAPGKQRELVAGIISGGSTNYADSVKGRLTISRDNAKNTVYLQMNSLKPEDTAVYYCSFVTTNSDYDLGRDYWGQGTQVTVSSGGGGSGGGSAVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSTSGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSEVQLVESGGGLVQAGGSLRLSCAASGSIFKVNAMGWYRQAPGKQRELVAGIISGGSTNYADSVKGRLTISRDNAKNTVYLQMNSLKPEDTAVYYCSFVTTNSDYDLGRDYWGQGTQVTVSSIL6R44, SEQ ID NO: 32EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYDIGWFRQAPGKEREGVSGISSSDGNTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTQVTVSSGGGGSGGGSAVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGFTFDDYDIGWFRQAPGKEREGVSGISSSDGNTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTQVTVSS IL6R49, SEQ ID NO: 33EVQLVESGGGLVQAGGSLRLSCAASGRTSSSYDMTWYRQVPGKEREFVAVISWSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCNAYTGGGDDYWGQGTQVTVSSGGGGSGGGSAVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTSSSYDMTWYRQVPGKEREFVAVISWSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCNAYTGGGDDYWGQGTQVTVSSIL6R53, SEQ ID NO: 34EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRATEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTQVTVSSGGGGSGGGSAVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRATEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCVKGSTAIVGVPPTYPDEYDYMGQGTQVTVSS

TABLE B-4 Protein sequences of sequence optimized NanobodiesIL6R61, SEQ ID NO: 40EVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRLTISRDNAKNTVYLQMNSLRPEDTAVYYCSFVTTNSDYDLGRDYWGQGTLVTVSSIL6R62, SEQ ID NO: 41EVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCSFVTTNSDYDLGRDYWGQGTLVTVSSIL6R63, SEQ ID NO: 42EVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRLTISRDNAKNTLYLQMNSLRPEDTAVYYCSFVTTNSDYDLGRDYWGQGTLVTVSSIL6R64, SEQ ID NO: 43EVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRLTISRDNAKNTVYLQMNSLRPEDTAVYYCAFVTTNSDYDLGRDYWGQGTLVTVSSIL6R65, SEQ ID NO: 44EVQLVESGGGLVQPGGSLRLSCAASGSIFKVNAMGWYRQAPGKGRELVAGIISGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFVTTNSDYDLGRDYWGQGTLVTVSSIL6R71, SEQ ID NO: 45EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISSDNAKNTVYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSSIL6R72, SEQ ID NO: 46EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISSDNAKNTVYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSSIL6R73, SEQ ID NO: 47EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRETISRDNAKNTVYLQMNSERPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGILVTVSSIL6R74, SEQ ID NO: 48EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISSDNAKNTLYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSSIL6R75, SEQ ID NO: 49EVQLVESGGGLVQPGGSLRLSCAASFTFSDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISRDNAKNTLYLQMSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSSIL6R81, SEQ ID NO: 50EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRATEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSIL6R82, SEQ ID NO: 51EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSIL6R83, SEQ ID NO: 52EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRATEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSIL6R84, SEQ ID NO: 53EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSIL6R85, SEQ ID NO: 54EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRGLEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSIL6R86, SEQ ID NO: 55EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRALEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSIL6R87, SEQ ID NC: 56EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKATEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSIL6R88, SEQ ID NO: 57EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGTEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSIL6R89, SEQ ID NO: 58EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGRGTEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSSIL6R90, SEQ ID NO: 59EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKALEWVSAISWNGNNTYYTESMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCVKGSTAIVGVPPTYPDEYDYWGQGTLVTVSS

TABLE B-5 Protein sequences of affinity matured NanobodiesPMP7F4, SEQ ID NO: 60EVQLVESGGGLVQPGGSLRLSCAASGTTFKVNVMAWYRQAPGKGRELVAGIINGGSTTYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFVTTNSDYDLGRDYWGQGTLVTVSSPMP7C4, SEQ ID NO: 61EVQLVESGGGLVQPGGSLRLSCAASGTTFRINVMAWYRQAPGKGRELVAGIITNGSTSYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFVTTNSDYDLGRDYWGQGTLVTVSSPMP7D6, SEQ ID NO: 62EVQLVESGGGLVQPGGSLRLSCAASGSIFRVNVMAWYRQAPGKGRELVAAVINGGTTTYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFVTTNSDYDLGRDYWGQGTLVTVSSPMP7G7, SEQ ID NO: 63EVQLVESGGGLVQPGGSLRLSCAASGTTFKINIMAWYRQAPGKGRELVAGVITGGNTTYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFVTTNSDYDLGRDYWGQGTLVTVSSPMP7G8, SEQ ID NO: 64EVQLVESGGGLVQPGGSLRLSCAASGSTFRINVMAWYRQAPGKGRELVAGVINDGSTTYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFVTTNSDYDLGRDYWGQGTLVTVSSPMP20F6, SEQ ID NO: 65EVQLVESGGGLVQPGGSLRLSCAASGSVFKINVMAWYRQAPGKGRELVAGIVSGGSTSYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFITTNSDYDLGRRYWGQGTLVTVSSPMP20A11, IL6R300, SEQ ID NO: 66EVQLVESGGGLVQPGGSLRLSCAASGSVFKINVMAWYRQAPGKGRELVAGIISGGSTSYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFITTESDYDLGRRYWGQGTLVTVSSPMP20E10, SEQ ID NO: 67EVQLVESGGGLVQPGGSLRLSCAASGSVFKINVMAWYRQAPGKGRELVAGIVSGGSTSYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFITTESDYDLGRRYWGQGTLVTVSSPMP21A10, SEQ ID NO: 68EVQLVESGGGLVQPGGSLRLSCAASGSIFKINVMAWYRQAPGKGRELVAGIVTGGSTSYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFITTESDYDLGRRYWGQGTLVTVSSPMP21D11, SEQ ID NO: 69EVQLVESGGGLVQPGGSLRLSCAASGSVFKINVMAWYRQAPGKGRELVAGIVTGGSTSYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFITTESDYDLGRRYWGQGTLVTVSS

TABLE B-6 Protein sequences of formatted affinity matured/sequence optimized Nanobodies IL6R304, SEQ ID NO: 70EVQLVESGGGLVQPGGSLRLSCAASGSVFKINVMAWYRQAPGKGRELVAGIISGGSTSYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFITTESDYDLGRRYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSIL6R305, SEQ ID NO: 71EVQLVESGGGLVQPGGSLRLSCAASGSVFKINVMAWYRQAPGKGRELVAGIISGGSTSYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFITTESDYDLGRRYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSVFKINVMAWYRQAPGKGRELVAGIISGGSTSYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFITTESDYDLGRRYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSS QGTLVTVSSIL6R306, SEQ ID NO: 72EVQLVESGGGLVQPGGSLRLSCAASGSVFKINVMAWYRQAPGKGRELVAGIISGGSTSYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFITTESDYDLGRRYWGQGTLVTVESGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSVFKINVMAWYRQAPGKGRELVAGIISGGSTSYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFITTESDYDLGRRYWG QGTLVTVSSIL6R202, SEQ ID NO: 73EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQG TLVTVSS

TABLE B-7 Preferred, but non-limiting examples of albumin-binding Nanobodies ALB-1, SEQ ID NO: 97AVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSALB-8(humanized ALB-1), SEQ ID NO: 98EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS ALB-2, SEQ ID NO: 99AVQLVESGGSLVQGGGSLRLACAASERIFDLNLMGWYRQGPGNERELVATCITVGDSTNYADSVKGRFTISMDYTKQTVYLHMNSLRPEDTGLYYCKIRRTWHSELWGQGTQVTVSS

TABLE B-8 Sequence listing of linkers GS30, SEQ ID NO: 101GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS GS15, SEQ ID NO: 102 GGGGSGGGGSGGGGSGS9, SEQ ID NO: 103 GGGGSGGGS GS7, SEQ ID NO: 104 SGGSGGSLlama upper long hinge region, SEQ ID NO: 105 EPKTPKPQPAAA

TABLE C-1 Materials used for the isolation of IL-6R binding NanobodiesSupplier Description Human IL-6 Diaclone Recombinant protein produced inE. coli Human bio-IL-6 Diaclone/PE Human IL-6 from Diacione biotinylatedby PE (6 biotins/molecule) Human soluble Peprotech Recombinant proteinproduced in IL-6R HEK293 cells (cat. # 200-06R) Human soluble R & DSystems G Recombinant protein produced IL-6R in Sf21 cells (cat. #227-SR) MAb BR-6 Diaclone Neutralizing anti-IL-6R MAb MAb BN-12 DiacloneNon-neutralizing anti-IL-6R MAb MAb M182 BD Biosciences Biotinylatedanti-IL-6R MAb Llama IgG (h & l) Bethyl Labs PAb against llama IgGraised in Antibody HRP goat conjugated TF-1 cell line ECACC J CellPhysiol 1989; 140: 323; no. 93022307 Exp Cell Res 1993: 208: 35

TABLE C-2 Immunization schedule Day Llama 081 Llama 082 Tissuecollection  0 100 μg 100 μg 10 ml pre-immune blood  7 100 μg 100 μg — 14 50 μg  50 μg — 21  50 μg  50 μg — 28  50 μg  50 μg 10 ml immune blood35  50 μg  50 μg — 39 150 ml immune blood PBL1 lymph node bow biopsy 43150 ml immune blood PBL2 52  50 μg  50 μg 59 100 ml immune blood NC1

TABLE C-3 Characteristics of the Nanobody libraries obtained from theimmunized llamas Library size % insert Llama 81 6 × 10⁷ 87 Llama 82 5 ×10⁷ 78

TABLE C-4 Conditions used for the selection of the NanobodiesImmobilization/ Concentration/ Elu- Method capture Antigen amount tionMagnetic Streptavidin bio-IL-6R 0, 1, 10, 100 ng Tryp- beads sinSolution Streptavidin bio-IL-6R 0, 0.01, 0.1, 1 nM Tryp- beads sin PlateBN-12 IL-6R 0, 1, 10, 100 nM Tryp- (Peprotech) sin Plate BN-12 IL-6R 0,1, 10, 100 nM Tryp- (R & D) sin

TABLE C-5 Screening statistics Number of Number of Number Number ofclones inhibitors of clones unique Assay screened (%) sequencedsequences IL-6-IL-6R 1536 72 (4.7%) 46 14

TABLE C-6 k_(off)-values of inhibitory Nanobodies Nanobody Nanobody IDID k_(off) (s⁻¹) PMP40H5 IL6R14 1.14E−04 PMP35E11 IL6R11 4.17E−04PMP32C9 IL6R04 1.50E−04 PMP35H4 IL6R13 1.78E−04 PMP32E10 IL6R05 1.27E−03PMP30C11 IL6R02 2.94E−03 PMP35C10 IL6R10 5.09E−04 PMP34G9 IL6R091.39E−03 PMP31A4 IL6R03 1.60E−03 PMP32E2 IL6R06 8.86E−04 PMP33A3 IL6R072.42E−04 PMP34A12 IL6R08 ND PMP28E11 IL6R01 ND PMP35F4 IL6R12 8.96E−04

TABLE C-7 Yields of Nanobody cell cultures Nanobody yield yield ID (mg)(mg/l) PMP40H5 0.14 0.6 PMP35E11 0.65 2.6 PMP32C9 0.33 6.5 PMP35H4 0.499.8 PMP32E10 0.78 3.1 PMP30C11 0.53 2.5 PMP35C10 0.53 2.1 PMP34G9 0.091.8 PMP31A4 1.06 4.2 PMP32E2 1.57 6.3 PMP33A3 0.33 1.3 PMP34A12 0.57 2.3PMP28E11 0.08 1.6 PMP35F4 0.24 1.0

TABLE C-8 IC50-values of selected Nanobodies Nanobody ID Sample IC50 (M)IL6R01 PMP28E11 7.26E−10 IL6R02 PMP30C11 1.69E−09 IL6R03 PMP31A47.16E−10 IL6R04 PMP32C9 9.30E−11 IL6R05 PMP32E10 6.26E−10 IL6R06 PMP32E21.21E−09 IL6R07 PMP33A3 1.44E−09 IL6R08 PMP34A12 1.18E−09 IL6R09 PMP34G92.38E−10 IL6R10 PMP35C10 5.96E−10 IL6R11 PMP35E11 1.58E−10 IL6R12PMP35F4 5.17E−10 IL6R13 PMP35H4 4.77E−11 IL6R14 PMP40H5 2.22E−10

TABLE C-9 Kinetic parameters for a selected subset of 14 inhibitoryanti-IL-6R Nanobodies Nanobody ID Nanobody ID k_(off) (s⁻¹) k_(on)(1/Ms) K_(d) (nM) IL6R01 PMP28E11 1.10E−04 2.62E+05 0.4 IL6R02 PMP30C112.94E−03 8.40E+05 5.9 4.95E−03 IL6R03 PMP31A4 1.47E−03 4.84E+05 3.01.60E−03 IL6R04 PMP32C9 9.42E−05 3.65E+05 0.3 1.50E−04 IL6R05 PMP32E101.41E−03 1.44E+05 9.8 1.27 E−03 IL6R06 PMP32E2 8.86E−04 1.07E+06 7.17.57E−03 IL6R07 PMP33A3 2.42E−04 ND ND IL6R08 PMP34A12 1.97E−03 1.94E+0510.2  IL6R09 PMP34G9 1.29E−03 6.41E+05 2.0 1.30E−03 1.11E+06 1.21.39E−03 IL6R10 PMP35C10 5.26E−04 4.14E+05 1.3 5.09E−04 IL6R11 PMP35E113.40E−04 3.91E+05 0.9 3.96E−04 2.15E+05 1.9 4.17E−04 IL6R12 PMP35F41.16E−03 6.78E+05 1.7 8.96E−04 IL6R13 PMP35H4 1.21E−04 2.31E+05 0.51.09E−04 1.37E+05 0.8 1.78E−04 IL6R14 PMP40H5 1.00E−04 4.02E+05 0.31.14E−04

TABLE C-10 IC50 values for Nanobody inhibition of XG-1 cellproliferation Nanobody ID IC50 (nM) IC50 (nM) + HSA IL6R01 ND IL6R0231.0 IL6R03 16.2 17.5 IL6R04 0.1 0.1 IL6R05 7.3 IL6R06 42.1 IL6R07 50.5IL6R08 36.6 IL6R09 2.7 3.0 IL6R10 2.5 IL6R11 5.4 IL6R12 2.8 IL6R13 1.41.3 IL6R14 0.6 0.8 Reference Fab 6.0

TABLE C-11 IC50 values for Nanobody inhibition of TF1 cell proliferationNanobody ID IC50 (nM) IL6R01 ND IL6R02 94.7 IL6R03 62.1 IL6R04 0.4IL6R05 38.0 IL6R06 137.9 IL6R07 374.9 IL6R08 24.3 IL6R09 8.7 IL6R10 9.9IL6R11 9.9 IL6R12 6.8 IL6R13 5.2 IL6R14 1.5 Reference Fab 9.2

TABLE C-12 Competition with the reference Fab for binding IL-6R asdetermined in an alphascreen assay Residual reference-Fab Nanobody IDbinding to IL-6R (%) IL6R01 49 IL6R02 86 IL6R03 5 IL6R04 50 IL6R05 64IL6R06 36 IL6R07 80 IL6R08 99 IL6R09 62 IL6R10 102 IL6R11 40 IL6R12 103IL6R13 25 IL6R14 96

TABLE C-13 Summary Nanobody characteristics IC50 IC50 IC50 Ref Fab K_(d)(nM) (nM) (nM) competition ID (nM) (IL-6/IL-6R) XG-1 TF-1 (%) IL6R01 0.40.73 ND ND 49 IL6R02 5.9 1.69 31.0 94.7 86 IL6R03 3.0 0.72 16.2 62.1 5IL6R04 0.3 0.09 0.1 0.4 50 IL6R05 9.8 0.63 7.3 38.0 64 IL6R06 7.1 1.2142.1 137.9 36 IL6R07 ND 1.44 50.5 374.9 80 IL6R08 10.2 1.18 36.6 24.3 99IL6R09 2.0 0.24 2.7 8.7 62 IL6R10 1.3 0.60 2.5 9.9 102 IL6R11 0.9 0.165.4 9.9 40 IL6R12 1.7 0.52 2.8 6.8 103 IL6R13 0.5 0.05 1.4 5.2 25 IL6R140.3 0.22 0.6 1.5 96

TABLE C-14 Nomenclature (ID) of formatted Nanobodies ID format SEQ ID NOIL6R22 PMP30C11-9GS-ALB1 19 IL6R23 PMP31A4-9GS-ALB1 20 IL6R24PMP32C9-9GS-ALB1 21 IL6R25 PMP32E10-9GS-ALB1 22 IL6R26 PMP32E2-9GS-ALB123 IL6R28 PMP34A12-9GS-ALB1 24 IL6R29 PMP34G9-9GS-ALB1 25 IL6R30PMP35C10-9GS-ALB1 26 IL6R31 PMP35E11-9GS-ALB1 27 IL6R32 PMP35F4-9GS-ALB128 IL6R33 PMP35H4-9GS-ALB1 29 IL6R34 PMP40H5-9GS-ALB1 30 IL6R43PMP31A4-9GS-ALB1-9GS-31A4 31 IL6R44 PMP32C9-9GS-ALB1-9GS-32C9 32 IL6R49PMP34G9-9GS-ALB1-9GS-34G9 33 IL6R53 PMP35H4-9GS-ALB1-9GS-35H4 34

TABLE C-15 Expression yields of bispecific anti-IL-6R NanobodiesNanobody yield yield ID ID (mg) (mg/l) PMP30C11 IL6R22 1.1 4.2 PMP31A4IL6R23 0.3 0.6 PMP32C9 IL6R24 0.5 2.0 PMP32E10 IL6R25 1.3 5.0 PMP32E2IL6R26 1.1 4.2 PMP34A12 IL6R28 2.1 8.4 PMP34G9 IL6R29 3.3 13.2 PMP35C10IL6R30 0.9 3.7 PMP35E11 IL6R31 1.8 7.3 PMP35F4 IL6R32 0.5 1.1 PMP35H4IL6R33 1.9 7.5 PMP40H5 IL6R34 0.4 0.9

TABLE C-16 IC50 values of bivalent Nanobodies in alphascreen competitionassay Nanobody ID IC50 (M) IL6R22 5.59E−10 IL6R24 1.45E−10 IL6R256.43E−10 IL6R26 1.67E−09 IL6R28 3.26E−10 IL6R29 1.23E−10 IL6R30 3.43E−10IL6R31 1.31E−10 IL6R32 2.68E−10 IL6R33 1.39E−10 IL6R34 1.46E−10reference-Fab 5.92E−10

TABLE C-17 IC50 values of formatted Nanobodies in the XG-1 proliferationassay ID IC50 (nM) IC50 (nM) + HSA IL6R22 50.2 IL6R23 16.9 90.4 IL6R240.2 0.5 IL6R25 8.4 IL6R26 65.3 IL6R28 4.4 IL6R29 3.6 13.4 IL6R30 27.2IL6R31 4.6 IL6R32 1.6 IL6R33 2.6 15.4 IL6R34 0.8 2.5 IL6R44 0.07 0.17IL6R49 0.06 0.19 IL6R53 0.13 0.61 Ref-IgG 0.47

TABLE C-18 Kinetic parameters of IL-6R binding Nanobody ID k_(d) (s⁻¹)k_(a) (1/Ms) K_(d) (nM) IL6R22 5.7E−03 3.3E+05 16.9 IL6R23 1.5E−033.2E+05 4.6 IL6R24 1.1E−04 3.7E+05 0.3 IL6R25 1.2E−03 1.2E+05 10.3IL6R26 6.9E−03 4.5E+05 15.5 IL6R28 5.3E−04 2.4E+05 2.2 IL6R29 1.5E−037.1E+05 2.1 IL6R30 1.2E−03 1.6E+05 7.5 IL6R31 3.8E−04 1.6E+05 2.3 IL6R321.3E−03 1.0E+06 1.3 IL6R33 1.25E−04  1.1E+05 1.1 IL6R34 1.1E−04 2.6E+050.4

TABLE C-19 K_(d) values of formatted Nanobodies for binding to serumalbumin from different species Human Mouse Cyno Rhesus Baboon ID K_(d)(nM) K_(d) (nM) K_(d) (nM) K_(d) (nM) K_(d) (nM) IL6R22 11.1 108 IL6R2316 275 27.6 23.8 23.2 IL6R24 15 122 28.3 28.3 40.3 IL6R25 13.9 122IL6R26 9.4 73 IL6R28 10.6 180 IL6R29 10.8 83 19 20.6 26.8 IL6R30 12.1113 IL6R31 13.4 86.8 IL6R32 10 179 IL6R33 27.3 98.6 24.5 24.6 32.3IL6R34 9.2 111 15 14.7 18.9 IL6R44 51.4 993 43 IL6R53 35 497 ALB-1 0.66.5

TABLE C-20 Kinetic parameters of sequence optimized variants of IL6R03,04 and 13 IL6R03 IL6R61 KD (nM) 2 (3.0 nM) ka (1/Ms) 8.50E+05 kd (1/s)1.70E−03 IL6R62 KD (nM) 2.2 ka (1/Ms) 9.29E+05 kd (1/s) 2.07E−03 IL6R63KD (nM) 3.7 ka (1/Ms) 9.90E+05 kd (1/s) 3.65E−03 IL6R64 KD (nM) ND ka(1/Ms) ND kd (1/s) 1.00E−03 IL6R04 IL6R71 KD (nM) 0.2 (0.3 nM) ka (1/Ms)7.03E+05 kd (1/s) 1.53E−04 IL6R72 KD (nM) 0.3 ka (1/Ms) 5.43E+05 kd(1/s) 1.80E−04 IL6R73 KD (nM) 0.3 ka (1/Ms) 6.98E+05 kd (1/s) 2.33E−04IL6R74 KD (nM) 0.2 ka (1/Ms) 7.67E+05 kd (1/s) 1.22E−04 IL6R13 IL6R81 KD(nM) 0.4 (0.7 nM) ka (1/Ms) 3.20E+05 kd (1/s) 1.28E−04 IL6R82 KD (nM)5.1 ka (1/Ms) 6.19E+05 kd (1/s) 3.14E−03 IL6R83 KD (nM) 0.3 ka (1/Ms)3.50E+05 kd (1/s) 1.20E−04 IL6R84 KD (nM) 5.4 ka (1/Ms) 7.62E+05 kd(1/s) 4.09E−03

TABLE C-21 k_(off) values of sequence optimized variants of IL6R13 IDk_(off) (s⁻¹) IL6R13 2.1E−04 IL6R85 2.1E−03 IL6R86 1.7E−03 IL6R871.1E−04 IL6R88 2.6E−04 IL6R89 1.9E−04 IL6R90 1.9E−03

TABLE C-22 Kinetic parameters of sequence optimized variants of IL6R03,04 and 13 IL6R03 IL6R65 KD (nM) 4 (3.0 nM) ka (1/Ms) 6.00E+05 kd (1/s)2.35E−03 IL6R04 IL6R75 KD (nM) 0.1 (0.3 nM) ka (1/Ms) 1.00E+06 kd (1/s)  1E−04 IL6R13 IL6R88 KD (nM) 0.9 (0.7 nM) ka (1/Ms) 2.30E+05 kd (1/s)2.13E−04

TABLE C-23 IC50 values of sequence optimized and WT Nanobodies in XG-1assay IC50 values (nM) Parental Sequence optimized IL6R03 17 IL6R65 26IL6R04 0.1 IL6R75 0.04 IL6R13 1.4 IL6R88 3.3

TABLE C-24 Kinetic parameters of Nanobody binding to cyno IL-6R ID k_(a)(1/Ms) k_(d) (1/s) K_(D) (nM) IL6R03 3.70E+05 1.64E−03 4.4 IL6R651.65E+05 1.97E−03 12 IL6R04 5.86E+04 1.00E−02 171 IL6R201* 2.18E+056.11E−03 28.1 *IL6R201 is the tagless version of IL6R75

TABLE C-25 Tm values of ILR65 and affinity matured IL-6R Nanobodies Tmfirst run Tm second run average Tm IL6R65 70.44 70.64 70.54 71.07 70.67PMP7F4 76.68 76.55 76.58 76.44 76.56 21A10 74.56 74.22 74.39 74.39 74.3920E10 75.29 75.22 75.31 75.38 75.30 20A11 74.42 74.03 74.12 74.19 74.1921D11 74.03 74.29 74.45 74.25 74.26 20F6 74.36 74.29 74.45 74.32 74.36

TABLE C-26 Kinetic parameters for affinity of IL6R65 and affinitymatured variants Nanobody k_(a) (M⁻¹.s⁻¹) k_(d) (s⁻¹) K_(d) (pM) IL6R651.0E+06  3.8E−03 3800 20E10 1.0E+06* 3.3E−05  33 21D11 1.0E+06* 3.5E−05 35 21A10 1.0E+06* 1.2E−05  12 20F6 1.0E+06* 3.3E−05  33 20A11 1.0E+06 1.9E−05  19 *estimated

TABLE C-27 Formats of affinity matured anti-IL6R Nanobodies Sequenceoptimized Format Nanobody Name

20A11-9GS-ALB8 IL6R304

20A11-9GS-20A11- 9GS-ALB8 IL6R305

20A11-9GS-ALB8- 9GS-20A11 IL6R306

TABLE C-28 Potency of formatted Nanobodies vs. references in TF-1proliferation assay at 100 IU/mL IL-6 Compound IC50 (nM) Stdev (nM)Repeats 20A11 0.283 0.256 3 IL6R304 0.715 0.390 2 IL6R305 0.098 0.046 4IL6R306 0.341 0.162 3 reference Fab 6.262 0.706 2 reference IgG 0.9210.275 4

TABLE C-29 Potency of formatted Nanobodies in TF-1 proliferation assayat 5000 IU/mL IL-6 Compound IC50 (nM) 20A11 15.22 IL6R304 15.48 IL6R305 5.49 IL6R306 23.19 Reference IgG 144.5 

TABLE C-30 IC50 values (nM) of formatted Nanobodies for neutralizationof IL-6 binding to sIL-6R in human plasma Compound IC50 at normal IL-6IC50 at high IL-6 Ratio (high/low) reference IgG 0.258 1.69  6.54IL6R20A11 0.198 0.356 1.80 IL6R304 0.229 0.634 2.77 IL6R305 0.137 0.3352.44 IL6R306 0.412 2.39  5.80

TABLE C-31 EC50 values (nM) for binding of the formatted affinitymatured Nanobodies to CHO 4D5 (4PL) Compound EC50 (nM) IL6R20A11 1.396 IL6R304 1.939  IL6R305 0.8984 IL6R306 6.154 

TABLE C-32 EC50 values (nM) for binding of the formatted affinitymatured Nanobodies to PBL from 2 donors Compound L1 L2 M1 M2 G1 G2IL6R20A11 3.65 2.924 3.621 4.777 2.415 5.614 IL6R304 9.273 20.68 8.24114.17 5.985 17.32 IL6R305 4.282 25.79 2.906 4.262 2.434 4.927 IL6R306 6049.59 49.38 59.34 53.99 77.68 L: lymphocytes, M: monocytes; G:granulocytes

TABLE C-33 Kinetic parameters for binding of formatted affinity maturedNanobodies to human and cyno serum albumin Human serum albumin Cynoserum albumin k_(a) k_(d) K_(D) k_(a) k_(d) K_(D) Nanobody (M⁻¹s⁻¹)(s⁻¹) (nM) (M⁻¹s⁻¹) (s⁻¹) (nM) ALB11 5.45E+05 1.68E−03 3.08 5.11E+051.53E−06 2.99 IL6R304 2.15E+05 4.75E−03 22.1 1.95E+05 4.56E−03 23.4IL6R305 2.01E+05 4.07E−03 20.3 2.03E+05 3.87E−03 19.1 IL6R306 2.25E+053.83E−03 17.1 2.12E+05 3.70E−03 17.4

TABLE C-34 Kinetic parameters for binding of the formatted affinitymatured Nanobodies to human and cyno IL-6R Human IL-6R Cyno IL-6R k_(a)k_(d) K_(D) k_(a) k_(d) K_(D) Nanobody (M⁻¹s⁻¹) (s⁻¹) (pM) (M⁻¹s⁻¹)(s⁻¹) (pM) IL6R300 1E+06 ND ND 1E+06 ND ND IL6R304 7E+05 ≦1E−05 * ≦148E+05 2E−05 25 * Detection limit of the instrument

TABLE C-35 Comparison of IC50 values (nM) for neutralization of bindingof hIL-6 to plasma sIL-6R in cyno and human plasma Test items IC50 inhuman IC50 in cyno Ratio (human/cyno) reference IgG 0.258 0.166  1.55IL6R20A11 0.198 0.117  1.69 IL6R304 0.229 0.137  1.67 IL6R305 0.1370.0791 1.73 IL6R306 0.412 0.321  1.29

TABLE C-36 Dose groups for in vivo PK/PD analysis of IL6R304 and IL6R305Nanobody dose IL-6 dose N and sex of Group Test item (mg/kg b.w., i.v.)(μg/kg b.w., s.c.) animals Reference no.  6 IL6R304  0.4 5 μg/kg, 2 m, 1f 11, 12, 13  7  2 once daily for 7 1 m, 2 f 14, 15, 16  8 10 days,starting 24 1 m, 1 f 17, 18  9 IL6R305  0.4 hours post 1 m, 2 f 19, 20,21 10  2 Nanobody 2 m, 1 f 22, 23, 24 11 10 administration 1 m, 1 f 25,26 12 Negative  0 2 m, 1 f 27, 28, 29 control 13 Positive  5 mg/kgreference 1 m, 2 f 30, 31, 32 control IgG

TABLE C-37 Sampling times for PK analysis Number of Group Sampling timesper animal samples Groups 6-13 Day 0: pre injection of Nanobody, and 5min, 30 352 min, 3 h, and 8 h post Nanobody injection Days 1, 2, 3, 4,5, 6, 7: pre injection of IL-6 Days 8, 14, 21 and 29

TABLE C-38 Basic PK parameters of IL6R304 after a single i.v. bolusadministration at 0.4 mg/kg in cynomolgus monkey IL6R304: IV 0.4 mg/kgCV Parameter Unit 11m 12m 13f Mean % Vss mL/kg 45.1 44.6 38.8 42.8 8 CLmL/day/kg 24.8 28.2 21.5 24.8 14 MRT day 1.81 1.58 1.80 1.73 8 t_(1/2)λz1 day 1.92 1.54 1.72 1.73 11 λz1 Lower day 1 1 1 1.00 0 λz1 Upper day4 4 5 4.33 13 R² t_(1/2) λz1 0.998 0.997 0.999 0.998 0 t_(1/2) λz2 day0.566 0.504 0.521 0.530 6 λz2 Lower day 5 4 5 4.67 12 λz2 Upper day 7 67 6.67 9 R² t_(1/2) λz2 0.967 1.00 0.938 0.968 3 AUClast day*μg/mL 16.014.1 18.6 16.2 14 AUCextrap % 0.372 0.365 0.325 0.354 7 AUCinf day*μg/mL16.1 14.2 18.6 16.3 14 AUCinf/D day*kg/mL 0.040 0.036 0.047 0.041 14

TABLE C-39 Basic PK parameters of IL6R304 after a single i.v. bolusadministration at 2 mg/kg in cynomolgus monkey. IL6R304: IV 2 mg/kg CVParameter Unit 14m 15f 16f Mean % Vss mL/kg 56.0 55.9 49.3 53.7 7 CLmL/day/kg 9.99 11.0 10.1 10.4 6 MRT day 5.60 5.06 4.86 5.17 7 t_(1/2)λz1 day 5.79 4.34 4.87 5.00 15 λz1 Lower day 2 2 2 2 0 λz1 Upper day 1414 14 14 0 R² t_(1/2) λz1 0.994 0.979 0.991 0.988 1 t_(1/2) λz2 day 1.511.52 1.30 1.44 9 λz2 Lower day 14 14 14 14 0 λz2 Upper day 21 21 21 21 0R² t_(1/2) λz2 1.00 1.00 1.00 1.00 0 t_(1/2) λz3 day 5.61 5.95 — 5.78 4λz3 Lower day 21 21 — 21 0 λz3 Upper day 29 29 — 29 0 R² t_(1/2) λz31.00 1.00 1.00 1.00 0 AUClast day*μg/mL 200 181 197 192 5 AUCextrap %0.295 0.247 0.090 0.211 51 AUCinf day*μg/mL 200 181 197 193 5 AUCinf/Dday*kg/mL 0.100 0.091 0.099 0.096 5 The terminal parameters for some ofthe animals were calculated with two data-points only (R² is 1 bydefault)

TABLE C-40 Basic PK parameters of IL6R304 after a single i.v. bolusadministration at 10 mg/kg in cynomolgus monkey IL6R304: IV 10 mg/kgParameter Unit 17 m 18 f Mean CV % Vss mL/kg 76.5 88.8 82.7 10 CLmL/day/kg 7.66 10.35 9.00 21 MRT day 10.0 8.57 9.29 11 t_(1/2) λz1 day7.15 6.08 6.61 11 λz1 Lower day 1 1 1  0 λz1 Upper day 29 29 29  0 R²t_(1/2) λz1 0.990 0.990 0.990  0 AUClast day * μg/mL 1230 932 1081 19AUCextrap % 5.79 3.50 4.64 35 AUCinf day * μg/mL 1306 966 1136 21AUCinf/D day * kg/mL 0.131 0.097 0.114 21

TABLE C-41 Basic PK parameters of IL6R305 after a single i.v. bolusadministration at 0.4 mg/kg in cynomolgus monkey. IL6R305: IV 0.4 mg/kgCV Parameter Unit 19m 20f 21f Mean % Vss mL/kg 59.2 72.5 63.9 65.2 10 CLmL/day/kg 33.5 38.0 36.0 35.8 6 MRT day 1.77 1.91 1.77 1.82 4 t_(1/2)λz1 day 1.79 1.25 1.89 1.64 21 λz1 Lower day 1 1 1 1 0 λz1 Upper day 5 54 4.67 12 R² t_(1/2) λz1 0.997 0.981 0.997 0.992 1 t_(1/2) λz2 day 0.446— 0.495 0.470 7 λz2 Lower day 5 — 5 5 0 λz2 Upper day 6 — 6 6 0 R²t_(1/2) λz2 1.00 — 1.00 1.00 0 AUClast day*μg/mL 11.84 9.84 11.02 10.9 9AUCextrap % 0.765 6.58 0.855 2.73 122 AUCinf day*μg/mL 11.9 10.5 11.111.2 6 AUCinf/D day*kg/mL 0.030 0.026 0.028 0.028 6 The terminalparameters for some of the animals were calculated with two data-pointsonly (R² is 1 by default)

TABLE C-42 Basic PK parameters of IL6R305 after a single i.v. bolusadministration at 2 mg/kg in cynomolgus monkey. IL6R305: IV 2 mg/kg CVParameter Unit 22m 23m 24f Mean % Vss mL/kg 27.5 28.0 30.4 28.6 5 CLmL/day/kg 5.81 5.30 6.68 5.93 12 MRT day 4.73 5.28 4.55 4.85 8 t_(1/2)λz1 day 4.26 4.56 4.04 4.29 6 λz1 Lower day 2 2 2 2 0 λz1 Upper day 1414 14 14 0 R² t_(1/2) λz1 0.985 0.954 0.986 0.975 2 t_(1/2) λz2 day 1.332.34 1.16 1.61 40 λz2 Lower day 14 14 14 14 0 λz2 Upper day 21 21 21 210 R² t_(1/2) λz2 1 1 1 1 0 AUClast day*μg/mL 344 374 299 339 11AUCextrap % 0.089 0.791 0.041 0.307 137 AUCinf day*μg/mL 344 377 299 34011 AUCinf/D day*kg/mL 0.172 0.189 0.150 0.170 11 The terminal parametersfor some of the animals were calculated with two data-points only (R² is1 by default)

TABLE C-43 Basic PK parameters of IL6R305 after a single i.v. bolusadministration at 10 mg/kg in cynomolgus monkey. The terminal parametersfor some of the animals were calculated with two data-points only (R² is1 by default) IL6R305: IV 10 mg/kg Parameter Unit 25 m 26 f Mean CV %Vss mL/kg 38.3 59.2 48.7 30 CL mL/day/kg 6.91 8.60 7.76 15 MRT day 5.546.88 6.21 15 t_(1/2) λz1 day 5.63 9.10 7.37 33 λz1 Lower day 2 2 2  0λz1 Upper day 14 14 14  0 R² t_(1/2) z1 0.941 0.968 0.955  2 t_(1/2) λz2day 1.26 1.16 1.21  6 λz2 Lower day 21 21 21  0 λz2 Upper day 29 29 29 0 R² t_(1/2) λz2 1.00 1.00 1.00  0 AUClast day * μg/mL 1447 1162 130515 AUCextrap % 0.008 0.009 0.008 11 AUCinf day * μg/mL 1447 1162 1305 15AUCinf/D day * kg/mL 0.145 0.116 0.130 15

TABLE C-44 Summary of the anti-IL6R304 and anti-IL6R305 antibodyappearance to full Nanobody IL6R304 Cyno ADA positive* IL6R305 Cyno ADApositive*  0.4 11 m  >7 days  0.4 19 m  >7 days mg/kg 12 m  >7 daysmg/kg 20 f  >7 days 13 f  >7 days 21 f  >7 days  2 14 m  >7 days  2 22m >14 days mg/kg 15 f >14 days mg/kg 23 m No ADA detected 16 f Noresults 24 f >14 days due to high predose values 10 17 m >14 days 10 25m >14 days mg/kg 18 f >14 days mg/kg 26 f >14 days *>7 days: TD 7Negative, ≧TD 14 Positive; >14 days: TD 14 Negative, ≧TD 21 Positive

TABLE C-45 Pharmacodynamic parameters of IL6R304 in the cynomolgusmonkey Parameter Estimate % CV K_(in) (ng/mL/h) 2.40 7.37 R₀ (ng/mL)21.4 5.23 I_(max) (%) 0.970 0.34 IC₅₀ (μg/mL) 0.146 15.8 IC₅₀ (nM) 5.23n 1.43 15.3

TABLE C-46 Summary of mean (±SD) key PK-parameters of IL6R304 in thecynomolgus monkey after a single i.v. bolus at 1, 5, 10, 25 or 100 mg/kg1 mg/kg 5 mg/kg 10 mg/kg 25 mg/kg 100 mg/kg Parameter Unit n Mean SD nMean SD n Mean SD n Mean SD n Mean SD C₀ μg/mL 3  26.2 3.8 3  108.7 24.03 196.5 30.8 3 562.3 66.4 1 2840.5 — AUC_(inf) μg · h/mL 2^(a) 2015 1902^(b) 12338 768 2 25983 440 3 89526 28466 1 540612 — t_(1/2) d 2^(a)1.81 0.00 2^(b) 5.01 0.76 2 6.37 0.19 3 6.24 0.76 1 8.9 — CL mL/h/kg2^(a) 0.50 0.04 2^(b) 0.41 0.02 2 0.39 0.01 3 0.30 0.11 1 0.18 — VzmL/kg 2^(a) 46.5 3.0 2^(b) 67.3 18.5 2 82.3 1.7 3 72.6 18.1 1 57.6 — DNμg · h/mL 2^(a) 2015 — 2^(b) 2468 — 2 2598 — 3 3581 — 1 5406 — AUC_(inf)DN: Dose-normalized to 1 mg/kg ^(a)Animal 3 excluded from descriptivestatistics as no target mediated clearance was observed: t_(1/2) = 4.3days ^(b)Animal 6 excluded from descriptive statistics as targetmediated clearance was observed: t_(1/2) = 2.2 days

TABLE C-47 Pharmacokinetic parameters of IL6R304 in cynomolgus monkey.Parameter Estimate % CV V_(c) (mL/kg) 45.6 5.23 V_(d) (mL/kg) 14.8 15.2V_(s) (mL/kg) 24.9 16.1 V_(dss) (mL/kg) 85.3 CL_(NON-IL6R) (mL/h/kg)0.237 3.39 CL_(d) (mL/h/kg) 0.0475 29.9 CL_(s) (mL/h/kg) 2.86 40.4V_(max) (μg/h/kg) 1.971 11.2 K_(m) (μg/mL) 0.714 30.4 CL_(IL6R)(mL/h/kg) 2.76

1. Amino acid sequence directed against IL-6R, that comprises one ormore stretches of amino acid residues chosen from the following: a) SEQID NO's: 80; or b) a stretch of amino acid residues that has no morethan 2, preferably no more than 1 amino acid difference with one of SEQID NO's: 80, provided that the amino acid sequence comprising saidstretch of amino acid residues binds IL-6R with about the same or ahigher affinity compared to the amino acid sequence comprising saidstretch of amino acid residues without the 2 or 1 amino acid difference,said affinity as measured by surface plasmon resonance; and/or c) SEQ IDNO's: 84, 89 and 91; or d) a stretch of amino acid residues that has nomore than 2, preferably no more than 1 amino acid difference with one ofSEQ ID NO's: 84, 89 and 91, provided that the amino acid sequencecomprising said stretch of amino acid residues binds IL-6R with aboutthe same or a higher affinity compared to the amino acid sequencecomprising said stretch of amino acid residues without the 2 or 1 aminoacid difference, said affinity as measured by surface plasmon resonance;and/or e) SEQ ID NO's: 93-94; or f) a stretch of amino acid residuesthat has no more than 2, preferably no more than 1 amino acid differencewith one of SEQ ID NO's: 93-94, provided that the amino acid sequencecomprising said stretch of amino acid residues binds IL-6R with aboutthe same or a higher affinity compared to the amino acid sequencecomprising said stretch of amino acid residues without the 1 or 2 aminoacid difference, said affinity as measured by surface plasmon resonance.2. Amino acid sequence according to claim 1, which comprises SEQ ID NO:80, SEQ ID NO: 84 and SEQ ID NO:
 93. 3. Amino acid sequence according toclaim 1 selected from the group consisting of: a) SEQ ID NO's: 65-69; b)a sequence that has no more than 2, preferably no more than 1 amino aciddifference in one, two or all of its CDRs with one of SEQ ID NO's 65-69,provided that the amino acid sequence with no more than 2, preferably nomore than 1 amino acid difference in one, two or all of its CDRs bindsIL-6R with about the same or a higher affinity compared to the bindingby the one of SEQ ID NO's 65-69, said affinity as measured by surfaceplasmon resonance; c) a sequence that has no more than 2, preferably nomore than 1 amino acid difference with one of SEQ ID NO's 65-69,provided that the amino acid sequence with no more than 2, preferably nomore than 1 amino acid difference with one of SEQ ID NO's 65-69 bindsIL-6R with about the same or a higher affinity compared to the bindingby the one of SEQ ID NO's 65-69, said affinity as measured by surfaceplasmon resonance.
 4. Compound or construct, that comprises oressentially consists of one or more amino acid sequences according toclaim 1, and optionally further comprises one or more other groups,residues, moieties or binding units, optionally linked via one or morelinkers.
 5. Compound or construct according to claim 4, in which saidone or more other groups, residues, moieties or binding units providethe compound or construct with increased half-life and in which said oneor more other groups, residues, moieties or binding units are chosenfrom the group consisting of domain antibodies, amino acid sequencesthat are suitable for use as a domain antibody, single domainantibodies, amino acid sequences that are suitable for use as a singledomain antibody, “dAb”'s, amino acid sequences that are suitable for useas a dAb, or Nanobodies that can bind to serum albumin (such as humanserum albumin) or a serum immunoglobulin (such as IgG).
 6. Compound orconstruct according to claim 4, selected from the following polypeptidesequences: a) SEQ ID NO's 70-72; b) a polypeptide sequence that has nomore than 2, preferably no more than 1 amino acid difference in one, twoor all of its CDRs of the invention with one of SEQ ID NO's: 70-72,provided that the polypeptide sequence with no more than 2, preferablyno more than 1 amino acid difference in one, two or all of its CDRs ofthe invention binds IL-6R with about the same or a higher affinitycompared to the binding by the one of SEQ ID NO's: 70-72, said affinityas measured by surface plasmon resonance; c) a polypeptide sequence thathas no more than 2, preferably no more than 1 amino acid difference withone of SEQ ID NO's: 70-72, provided that the amino acid sequence with nomore than 2, preferably no more than 1 amino acid difference with one ofSEQ ID NO's: 70-72 binds IL-6R with about the same or a higher affinitycompared to the binding by the one of SEQ ID NO's: 70-72, said affinityas measured by surface plasmon resonance.
 7. Compound or constructaccording to claim 4, that has or essentially consists of the amino acidsequence of SEQ ID NO: 70 or the amino acid sequence of SEQ ID NO: 71.8. Compound or construct according to claim 4, that specifically bindsto hIL-6R with a dissociation constant (KD) of 1 nM to 1 pM moles/litreor less, preferably 500 pM to 1 pM moles/litre or less, more preferably100 pM to 1 pM moles/litre or less, or even more preferably about 50 pMto 1 pM or less.
 9. Monovalent construct, comprising or essentiallyconsisting of one amino acid sequence according to claim
 1. 10. Methodfor the preparation of a multivalent compound or construct, comprisingthe linking of an amino acid sequence according to claim 1 or amonovalent construct comprising or essentially consisting of one aminoacid sequence according to claim 1 to one or more groups, residues,moieties or binding units.
 11. Nucleic acid or nucleotide sequence, thatencodes an amino acid sequence according to claim 1, or a monovalentconstruct comprising or essentially consisting of one amino acidsequence according to claim 1, said nucleic acid optionally in the formof a genetic construct.
 12. Host or host cell that comprises a nucleicacid or nucleotide sequence or a genetic construct according to claim11.
 13. Method for producing an amino acid, said method at leastcomprising the steps of: a) expressing, in a suitable host cell or hostorganism or in another suitable expression system, a nucleic acid ornucleotide sequence or a genetic construct according to claim 11;optionally followed by: b) isolating and/or purifying the amino acidsequence thus obtained.
 14. Composition, such as a pharmaceuticalcomposition, comprising at least one amino acid sequence according toclaim 1, or a monovalent construct comprising or essentially consistingof one amino acid sequence according to claim
 1. 15. Method for theprevention and/or treatment of at least one of the diseases anddisorders associated with IL-6, with IL-6R, with the IL-6/IL-6R complexand/or with the signalling pathways and/or the biological functions andresponses in which IL-6, IL-6R and/or the IL-6/IL-6R complex areinvolved chosen from the group consisting of sepsis, various forms ofcancer, bone resorption, osteoporosis, cachexia, psoriasis, mesangialproliferative glomerulonephritis, Kaposi's sarcoma, AIDS-relatedlymphoma, and inflammatory diseases, said method comprisingadministering, to a subject in need thereof, a pharmaceutically activeamount of at least one amino acid sequence according to claim 1, or amonovalent construct comprising or essentially consisting of one aminoacid sequence according to claim 1.